Quantitative autoradiography of the binding sites for [125I] iodoglyburide, a novel high-affinity ligand for ATP-sensitive potassium channels in rat brain

International Nuclear Information System (INIS)

Gehlert, D.R.; Gackenheimer, S.L.; Mais, D.E.; Robertson, D.W.

1991-01-01

We have developed a high specific activity ligand for localization of ATP-sensitive potassium channels in the brain. When brain sections were incubated with [ 125 I]iodoglyburide (N-[2-[[[(cyclohexylamino)carbonyl]amino]sulfonyl]ethyl]-5- 125 I-2- methoxybenzamide), the ligand bound to a single site with a KD of 495 pM and a maximum binding site density of 176 fmol/mg of tissue. Glyburide was the most potent inhibitor of specific [ 125 I]iodoglyburide binding to rat forebrain sections whereas iodoglyburide and glipizide were slightly less potent. The binding was also sensitive to ATP which completely inhibited binding at concentrations of 10 mM. Autoradiographic localization of [ 125 I]iodoglyburide binding indicated a broad distribution of the ATP-sensitive potassium channel in the brain. The highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain. These results indicate that the ATP-sensitive potassium channel has a broad presence in the rat brain and that a few select brain regions are enriched in this subtype of neuronal potassium channels

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

Science.gov (United States)

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-09-08

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.

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Dey, Sanjay; Biswas, Maitree; Sen, Udayaditya; Dasgupta, Jhimli

2015-04-03

Bacterial enhancer-binding proteins (bEBPs) oligomerize through AAA(+) domains and use ATP hydrolysis-driven energy to isomerize the RNA polymerase-σ(54) complex during transcriptional initiation. Here, we describe the first structure of the central AAA(+) domain of the flagellar regulatory protein FlrC (FlrC(C)), a bEBP that controls flagellar synthesis in Vibrio cholerae. Our results showed that FlrC(C) forms heptamer both in nucleotide (Nt)-free and -bound states without ATP-dependent subunit remodeling. Unlike the bEBPs such as NtrC1 or PspF, a novel cis-mediated "all or none" ATP binding occurs in the heptameric FlrC(C), because constriction at the ATPase site, caused by loop L3 and helix α7, restricts the proximity of the trans-protomer required for Nt binding. A unique "closed to open" movement of Walker A, assisted by trans-acting "Glu switch" Glu-286, facilitates ATP binding and hydrolysis. Fluorescence quenching and ATPase assays on FlrC(C) and mutants revealed that although Arg-349 of sensor II, positioned by trans-acting Glu-286 and Tyr-290, acts as a key residue to bind and hydrolyze ATP, Arg-319 of α7 anchors ribose and controls the rate of ATP hydrolysis by retarding the expulsion of ADP. Heptameric state of FlrC(C) is restored in solution even with the transition state mimicking ADP·AlF3. Structural results and pulldown assays indicated that L3 renders an in-built geometry to L1 and L2 causing σ(54)-FlrC(C) interaction independent of Nt binding. Collectively, our results underscore a novel mechanism of ATP binding and σ(54) interaction that strives to understand the transcriptional mechanism of the bEBPs, which probably interact directly with the RNA polymerase-σ(54) complex without DNA looping. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

Science.gov (United States)

Liao, Jielou

2004-03-01

Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

Interaction between nucleotide binding sites on chloroplast coupling factor 1 during ATP hydrolysis

Energy Technology Data Exchange (ETDEWEB)

Leckband, D.; Hammes, G.G.

1987-04-21

The initial hydrolysis of radioactively-labelled CaATP by chloroplast coupling factor 1 was studied with the quenched-flow method. The time course of hydrolysis can be described as a first-order conversion of the enzyme to an active form followed by steady-state formation of product. The rate constant for the first-order process is independent of substrate concentration but increased hyperbolically to a limiting value of 0.43 s/sup -1/ with increasing concentrations of free Ca/sup 2 +/. A mechanism involving a Ca/sup 2 +/-triggered conversion to an active form of the enzyme is consistent with the data. The steady-state rate varied sigmoidally with the CaATP concentration. Initial exchange of tightly bound ADP is complex: approx. 50% of the bound nucleotide is lost within 30 s, with complete exchange requiring several minutes. The first-order rate constant characterizing the rapid phase of the reaction increases hyperbolically to a limiting value of 0.26 s/sup -1/ as the concentration of CaATP is increased, indicating that the binding of CaATP to the enzyme promotes the exchange process. Modification of the quenched-flow apparatus permitted measurement of the rate of nucleotide exchange during steady-state catalysis. The value of the first-order rate constant characterizing this process is similar to the catalytic rate constant determined under identical conditions. When MgATP is tightly bound to the enzyme, none of the kinetic properties of the enzyme described above were significantly changes. The results obtained suggest a mechanism in which two sites on the enzyme participate in catalysis. Several possible mechanisms consistent with the data are discussed.

Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

KAUST Repository

Beke-Somfai, T.; Lincoln, P.; Norden, B.

2013-01-01

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

Rate of hydrolysis in ATP synthase is fine-tuned by  -subunit motif controlling active site conformation

KAUST Repository

Beke-Somfai, T.

2013-01-23

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F(o)F(1) ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F(1) performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.

Identification of an allosteric binding site for RORγt inhibition

Energy Technology Data Exchange (ETDEWEB)

Scheepstra, Marcel; Leysen, Seppe; vanAlmen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc (Merck); (Eindhoven)

2015-12-07

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

Long-Range Effects of Na(+) Binding in Na,K-ATPase Reported by ATP.

Science.gov (United States)

Middleton, David A; Fedosova, Natalya U; Esmann, Mikael

2015-12-01

This paper addresses the question of long-range interactions between the intramembranous cation binding sites and the cytoplasmic nucleotide binding site of the ubiquitous ion-transporting Na,K-ATPase using (13)C cross-polarization magic-angle spinning (CP-MAS) solid-state nuclear magnetic resonance. High-affinity ATP binding is induced by the presence of Na(+) as well as of Na-like substances such as Tris(+), and these ions are equally efficient promoters of nucleotide binding. CP-MAS analysis of bound ATP with Na,K-ATPase purified from pig kidney membranes reveals subtle differences in the nucleotide interactions within the nucleotide site depending on whether Na(+) or Tris(+) is used to induce binding. Differences in chemical shifts for ATP atoms C1' and C5' observed in the presence of Na(+) or Tris(+) suggest alterations in the residues surrounding the bound nucleotide, hydrogen bonding, and/or conformation of the ribose ring. This is taken as evidence of a long-distance communication between the Na(+)-filled ion sites in the membrane interior and the nucleotide binding site in the cytoplasmic domain and reflects the first conformational change ultimately leading to phosphorylation of the enzyme. Stopped-flow fluorescence measurements with the nucleotide analogue eosin show that the dissociation rate constant for eosin is larger in Tris(+) than in Na(+), giving kinetic evidence of the difference in structural effects of Na(+) and Tris(+). According to the recent crystal structure of the E1·AlF4(-)·ADP·3Na(+) form, the coupling between the ion binding sites and the nucleotide side is mediated by, among others, the M5 helix.

EPR study of manganese(II) binding to 55'-ATP, hemoglobin, and hemocyanin

Energy Technology Data Exchange (ETDEWEB)

Chang, S.S. (Duquesne Univ., Pittsburgh); Li, N.C.; Pratt, D.W.

1975-01-01

Several divalent metal ions affect the oxygen affinity of hemoglobin and hemocyanin. It is important, therefore, to understand the nature of metal-ion binding to these proteins. By comparing the EPR spectra of Mn(II), 0.001 M, in the absence and presence of carboxyhemoglobin or Limulus oxyhemocyanin (pH 7.3, Trizma buffer), the number of Mn binding sites, n, and the binding constant, K, can be determined. For carboxyhemoglobin, HbCO, we find 0.5 Mn binding sites per heme, K = 450 M/sup -1/. Each hemoglobin tetramer therefore binds two manganous ions suggesting that Mn(II), like Cu(II), may bind preferentially to one of the two types of subunits in hemoglobin. For hemocyanin, HcO/sub 2/, we find n = 5.8, K = 1.55 x 10/sup 3/ M/sup -1/. Each oxyhemocyanine therefore binds approximately six manganous ions, and the binding constant is three times larger than that for HbCO. We have also carried out similar experiments on 5'-ATP, and on solutions of HbCO and ATP containing McCl/sub 2/ or ZnCl/sub 2/. Zn(II) effectively competes with Mn(II) in binding hemoglobin and ATP, whereas Mg(II) does not, in accord with expectations from data on oxygen affinity of hemoglobin. (auth)

The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

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Hao Ding

Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

International Nuclear Information System (INIS)

Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie; Archambault, Jacques; Bullock, Peter A.

2011-01-01

With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner

A conserved mechanism of autoinhibition for the AMPK kinase domain: ATP-binding site and catalytic loop refolding as a means of regulation

International Nuclear Information System (INIS)

Littler, Dene R.; Walker, John R.; Davis, Tara; Wybenga-Groot, Leanne E.; Finerty, Patrick J. Jr; Newman, Elena; Mackenzie, Farell; Dhe-Paganon, Sirano

2010-01-01

A 1.9 Å resolution crystal structure of the isolated kinase domain from the α2 subunit of human AMPK, the first from a multicellular organism, is presented. The AMP-activated protein kinase (AMPK) is a highly conserved trimeric protein complex that is responsible for energy homeostasis in eukaryotic cells. Here, a 1.9 Å resolution crystal structure of the isolated kinase domain from the α2 subunit of human AMPK, the first from a multicellular organism, is presented. This human form adopts a catalytically inactive state with distorted ATP-binding and substrate-binding sites. The ATP site is affected by changes in the base of the activation loop, which has moved into an inhibited DFG-out conformation. The substrate-binding site is disturbed by changes within the AMPKα2 catalytic loop that further distort the enzyme from a catalytically active form. Similar structural rearrangements have been observed in a yeast AMPK homologue in response to the binding of its auto-inhibitory domain; restructuring of the kinase catalytic loop is therefore a conserved feature of the AMPK protein family and is likely to represent an inhibitory mechanism that is utilized during function

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.

Science.gov (United States)

Kasho, Kazutoshi; Fujimitsu, Kazuyuki; Matoba, Toshihiro; Oshima, Taku; Katayama, Tsutomu

2014-12-01

In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of an allosteric binding site for RORγt inhibition

NARCIS (Netherlands)

Scheepstra, M.; Leysen, S.; van Almen, G.; Miller, J.R.; Piesvaux, J.; Kutilek, V.; van Eenennaam, H.; Zhang, H.; Barr, K.; Nagpal, S.; Soisson, S.M.; Kornienko, M.; Wiley, K.; Elsen, N.; Sharma, S.; Correll, C.C.; Trotter, B.W.; Stelt, van der M.; Oubrie, A.; Ottmann, C.; Parthasarathy, G.; Brunsveld, L.

2015-01-01

RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of

Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels.

Science.gov (United States)

Wu, Jing-Xiang; Ding, Dian; Wang, Mengmeng; Kang, Yunlu; Zeng, Xin; Chen, Lei

2018-06-01

ATP-sensitive potassium channels (K ATP ) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K ATP channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K ATP channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.

Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

Energy Technology Data Exchange (ETDEWEB)

Shyy, Y.J.; Tian, G.; Tsai, M.D.

1987-10-06

Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

Directory of Open Access Journals (Sweden)

Li Wang

Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump.

Science.gov (United States)

Tejral, Gracian; Sopko, Bruno; Necas, Alois; Schoner, Wilhelm; Amler, Evzen

2017-01-01

Hydrolysis of ATP by Na + /K + -ATPase, a P-Type ATPase, catalyzing active Na + and K + transport through cellular membranes leads transiently to a phosphorylation of its catalytical α -subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp 369 to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ -phosphate group of ATP to the Asp 369 is achieved, analogous molecular modeling of the M 4 -M 5 loop of ATPase was performed using the crystal data of Na + /K + -ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr 338 and Ile 760 of the α 2 -subunit of Na + /K + -ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe 475 in the N-domain, the other one close to Asp 369 in the P-domain. However, binding of Mg 2+ •ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp 369 . Additional conformations of the cytoplasmic loop were found wobbling between "open conformation"  "semi-open conformation  "closed conformation" in the absence of 2Mg 2+ •ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg 543 and Asp 611 -triggers by binding of 2Mg 2+ •ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp 369 in the P-domain, and hence the start of Na + /K + -activated ATP hydrolysis.

[Adenylate cyclase from rabbit heart: substrate binding site].

Science.gov (United States)

Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

1981-08-01

The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM).

Science.gov (United States)

Chue, B; Ferguson, T A; Beaman, K D; Rosenman, S J; Cone, R E; Flood, P M; Green, D R

1989-01-01

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

Science.gov (United States)

Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

2018-02-01

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Hyperactivity of the Arabidopsis cryptochrome (cry1) L407F mutant is caused by a structural alteration close to the cry1 ATP-binding site.

Science.gov (United States)

Orth, Christian; Niemann, Nils; Hennig, Lars; Essen, Lars-Oliver; Batschauer, Alfred

2017-08-04

Plant cryptochromes (cry) act as UV-A/blue light receptors. The prototype, Arabidopsis thaliana cry1, regulates several light responses during the life cycle, including de-etiolation, and is also involved in regulating flowering time. The cry1 photocycle is initiated by light absorption by its FAD chromophore, which is most likely fully oxidized (FAD ox ) in the dark state and photoreduced to the neutral flavin semiquinone (FADH°) in its lit state. Cryptochromes lack the DNA-repair activity of the closely related DNA photolyases, but they retain the ability to bind nucleotides such as ATP. The previously characterized L407F mutant allele of Arabidopsis cry1 is biologically hyperactive and seems to mimic the ATP-bound state of cry1, but the reason for this phenotypic change is unclear. Here, we show that cry1 L407F can still bind ATP, has less pronounced photoreduction and formation of FADH° than wild-type cry1, and has a dark reversion rate 1.7 times lower than that of the wild type. The hyperactivity of cry1 L407F is not related to a higher FADH° occupancy of the photoreceptor but is caused by a structural alteration close to the ATP-binding site. Moreover, we show that ATP binds to cry1 in both the dark and the lit states. This binding was not affected by cry1's C-terminal extension, which is important for signal transduction. Finally, we show that a recently discovered chemical inhibitor of cry1, 3-bromo-7-nitroindazole, competes for ATP binding and thereby diminishes FADH° formation, which demonstrates that both processes are important for cry1 function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Patterns of predicted T-cell epitopes associated with antigenic drift in influenza H3N2 hemagglutinin.

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E Jane Homan

Full Text Available Antigenic drift allowing escape from neutralizing antibodies is an important feature of transmission and survival of influenza viruses in host populations. Antigenic drift has been studied in particular detail for influenza A H3N2 and well defined antigenic clusters of this virus documented. We examine how host immunogenetics contributes to determination of the antibody spectrum, and hence the immune pressure bringing about antigenic drift. Using uTOPE™ bioinformatics analysis of predicted MHC binding, based on amino acid physical property principal components, we examined the binding affinity of all 9-mer and 15-mer peptides within the hemagglutinin 1 (HA1 of 447 H3N2 virus isolates to 35 MHC-I and 14 MHC-II alleles. We provide a comprehensive map of predicted MHC-I and MHC-II binding affinity for a broad array of HLA alleles for the H3N2 influenza HA1 protein. Each HLA allele exhibited a characteristic predicted binding pattern. Cluster analysis for each HLA allele shows that patterns based on predicted MHC binding mirror those described based on antibody binding. A single amino acid mutation or position displacement can result in a marked difference in MHC binding and hence potential T-helper function. We assessed the impact of individual amino acid changes in HA1 sequences between 10 virus isolates from 1968-2002, representative of antigenic clusters, to understand the changes in MHC binding over time. Gain and loss of predicted high affinity MHC-II binding sites with cluster transitions were documented. Predicted high affinity MHC-II binding sites were adjacent to antibody binding sites. We conclude that host MHC diversity may have a major determinant role in the antigenic drift of influenza A H3N2.

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

International Nuclear Information System (INIS)

Penefsky, H.S.

1985-01-01

Incubation of [gamma- 32 P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma- 32 P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma- 32 P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide

Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

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Gracian Tejral

2017-03-01

Full Text Available Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation”  “semi-open conformation  “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

Energy Technology Data Exchange (ETDEWEB)

Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.; Steussy, Calvin Nicklaus; Stauffacher, Cynthia V. (Purdue)

2017-10-12

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

Science.gov (United States)

Tan, M P; Dolton, G M; Gerry, A B; Brewer, J E; Bennett, A D; Pumphrey, N J; Jakobsen, B K; Sewell, A K

2017-01-01

CD4 + T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4 + T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4 + T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4 + cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4 + and CD8 + T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4 + T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4 + T cells than CD8 + T cells. These results indicate that the CD4 + T cell component of current adoptive therapies using TCRs optimized for CD8 + T cells is below par and that there is room for substantial improvement. © 2016 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.

Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

International Nuclear Information System (INIS)

Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

1988-01-01

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

Identification of a peptide binding protein that plays a role in antigen presentation

International Nuclear Information System (INIS)

Lakey, E.K.; Margoliash, E.; Pierce, S.K.

1987-01-01

The helper T-cell response to globular proteins appears, in general, to require intracellular processing of the antigen, such that a peptide fragment containing the T-cell antigenic determinant is released and transported to and held on the surface of an Ia-expressing, antigen-presenting cell. However, the molecular details underlying these phenomena are largely unknown. The means by which antigenic peptides are anchored on the antigen-presenting cell surface was investigated. A cell surface protein is identified that was isolated by it ability to bind to a 24-amino acid peptide fragment of pigeon cytochrome c, residues 81-104, containing the major antigenic determinant for B10.A mouse T cells. This peptide binding protein, purified from [ 35 S]methionine-labeled cells, appears as two discrete bands of ≅72 and 74 kDa after NaDodSO 4 /PAGE. The protein can be eluted from the peptide affinity column with equivalent concentrations of either the antigenic pigeon cytochrome c peptide or the corresponding nonantigenic peptide of mouse cytochrome c. However, it does not bind to the native cytochromes c, either of pigeon or mouse, and thus the protein appears to recognize some structure available only in the free peptides. This protein plays a role in antigen presentation. Its expression is not major histocompatibility complex-restricted in that the blocking activity of the antisera can be absorbed on spleen cells from mice of different haplotypes. This peptide binding protein can be isolated from a variety of cell types, including B cells, T cells, and fibroblasts. The anchoring of processed peptides on the cell surface by such a protein may play a role in antigen presentation

Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

International Nuclear Information System (INIS)

Rebelo, Tânia S.C.R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M.H.; Noronha, João P.; Sales, M. Goreti F.

2014-01-01

Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10 −11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

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Wang Pu

2010-10-01

Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with SV40 T-antigen mutants defective in RB and P53 binding domains

International Nuclear Information System (INIS)

LingNah Su; Little, J.B.

1992-01-01

A series of human diploid fibroblast cell clones were developed by DNA transfection with either wild-type SV40 T-antigen (SV40T) or T-antigen mutants defective in its various functional domains. Cell clones expressing the wild-type SV40 T were significantly radioresistant as compared with clones transfected with the neo gene only (D o 192 ± 13 vs 127 ± 19). This radioresistance persisted in post-crisis, immortalized cell lines. A series of mutants with point or deletion mutations within each functionally active domain of SV40 T were also examined for their ability to alter radiosensitivity and induce morphological transformation. Cell clones transfected with T-antigen mutants defective in nuclear localization or origin binding showed increased radioresistance similar to clones transfected with wild-type T-antigen, and expressed morphological changes characteristic of SV40 T-transfected cells. (author)

Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

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Nandhitha Subramanian

Full Text Available The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.

Eight amino acids form the ATP recognition site of Na(+)/K(+)-ATPase

Czech Academy of Sciences Publication Activity Database

Kubala, Martin; Teisinger, Jan; Ettrich, Rüdiger; Hofbauerová, Kateřina; Kopecký ml., Vladimír; Baumruk, V.; Krumscheid, R.; Plášek, J.; Schoner, W.; Amler, Evžen

2003-01-01

Roč. 42, č. 21 (2003), s. 6446-6452 ISSN 0006-2960 R&D Projects: GA ČR GA204/01/0254; GA ČR GA204/01/1001; GA ČR GA309/02/1479 Grant - others:GA-(CZ) CZE00/033 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 123100001; CEZ:MSM 113100001; CEZ:MSM 113200001 Keywords : sodium pump * ATP-binding site * TNP-ATP Subject RIV: BO - Biophysics Impact factor: 3.922, year: 2003

Antigen-specific T cell activation independently of the MHC: chimeric antigen receptor (CAR-redirected T cells.

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Hinrich eAbken

2013-11-01

Full Text Available Adoptive T cell therapy has recently shown powerful in initiating a lasting anti-tumor response with spectacular therapeutic success in some cases. Specific T cell therapy, however, is limited since a number of cancer cells are not recognized by T cells due to various mechanisms including the limited availability of tumor-specific T cells and deficiencies in antigen processing or major histocompatibility complex (MHC expression of cancer cells. To make adoptive cell therapy applicable for the broad variety of cancer entities, patient's T cells are engineered ex vivo with pre-defined specificity by a recombinant chimeric antigen receptor (CAR which consists in the extracellular part of an antibody-derived domain for binding with a tumor-associated antigen and in the intracellular part of a TCR-derived signaling moiety for T cell activation. The specificity of CAR mediated T cell recognition is defined by the antibody domain, is independent of MHC presentation and can be extended to any target for which an antibody is available. We discuss the advantages and limitations of MHC-independent T cell targeting by an engineered CAR and review most significant progress recently made in early stage clinical trials to treat cancer.

Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A.

Science.gov (United States)

Cole, C N; Tornow, J; Clark, R; Tjian, R

1986-01-01

The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627-amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional

The mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase studied by molecular dynamics simulations

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Mykuliak V. V.

2014-03-01

Full Text Available Aim. To study the mechanisms of substrates interaction with the active site of Mycobacterium tuberculosis tyrosyl-tRNA synthetase (MtTyrRS. Methods. Complexes of MtTyrRS with tyrosine, ATP and tyrosyl adenylate were constructed by superposition of the MtTyrRS structure and crystallographic structures of bacterial TyrRS. All complexes of MtTyrRS with substrates were investigated by molecular dynamics (MD simulations in solution. Results. It was shown the formation of network of hydrogen bonds between substrates and the MtTyrRS active center, which were stable in the course of MD simulations. ATP binds in the active site both by hydrogen bonds and via electrostatic interactions with Lys231 and Lys234 of catalytic KFGKS motif. Conclusions. The L-tyrosine binding site in the enzyme active site is negatively charged, whereas the ATP binding site contains positive Lys231 and Lys234 residues of catalytic KFGKS motif. The occupancy of H-bonds between substrates and the enzyme evidences a significant conformational mobility of the active site.

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

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Shayla K Shorter

Full Text Available T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL, have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4 are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

A casein-kinase-2-related protein kinase is tightly associated with the large T antigen of simian virus 40

DEFF Research Database (Denmark)

Götz, C; Koenig, M G; Issinger, O G

1995-01-01

by the addition of protein kinase CK2 suggest that at least one of the T-antigen-associated protein kinases is CK2 or a protein-kinase-CK2-related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature-sensitive SV40-transformed......The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we...... show that immuno-purified T antigen from SV40-transformed cells and from baculovirus-infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation...

Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein.

Science.gov (United States)

Sakato, Miho; King, Stephen M

2003-10-31

The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.

Measurement of T-lymphocyte responses in whole-blood cultures using newly synthesized DNA and ATP.

Science.gov (United States)

Sottong, P R; Rosebrock, J A; Britz, J A; Kramer, T R

2000-03-01

The proliferative response is most frequently determined by estimating the amount of [(3)H]thymidine incorporated into newly synthesized DNA. The [(3)H]thymidine procedure requires the use of radioisotopes as well as lengthy periods of incubation (>72 h). An alternative method of assessing T-lymphocyte activation in whole-blood cultures involves the measurement of the nucleotide ATP instead of [(3)H]thymidine incorporation. In addition, the Luminetics assay of T-cell activation measures specific T-lymphocyte subset responses through the use of paramagnetic particles coated with monoclonal antibodies against CD antigens. This assay permits rapid (24 h) analysis of lymphocyte subset activation responses to mitogens and recall antigens in small amounts of blood.

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

Science.gov (United States)

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-07-01

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

Directory of Open Access Journals (Sweden)

Pene-Dumitrescu Teodora

2012-03-01

Full Text Available Abstract Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus.

Science.gov (United States)

Yamasaki, Takashi; Nakazaki, Yosuke; Yoshida, Masasuke; Watanabe, Yo-hei

2011-07-01

ClpB, a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), forms a ring-shaped hexamer and cooperates with the DnaK chaperone system to reactivate aggregated proteins in an ATP-dependent manner. The ClpB protomer consists of an N-terminal domain, an AAA+ module (AAA-1), a middle domain, and a second AAA+ module (AAA-2). Each AAA+ module contains highly conserved WalkerA and WalkerB motifs, and two arginines (AAA-1) or one arginine (AAA-2). Here, we investigated the roles of these arginines (Arg322, Arg323, and Arg747) of ClpB from Thermus thermophilus in the ATPase cycle and chaperone function by alanine substitution. These mutations did not affect nucleotide binding, but did inhibit the hydrolysis of the bound ATP and slow the threading of the denatured protein through the central pore of the T. thermophilus ClpB ring, which severely impaired the chaperone functions. Previously, it was demonstrated that ATP binding to the AAA-1 module induced motion of the middle domain and stabilized the ClpB hexamer. However, the arginine mutations of the AAA-1 module destabilized the ClpB hexamer, even though ATP-induced motion of the middle domain was not affected. These results indicated that the three arginines are crucial for ATP hydrolysis and chaperone activity, but not for ATP binding. In addition, the two arginines in AAA-1 and the ATP-induced motion of the middle domain independently contribute to the stabilization of the hexamer. © 2011 The Authors Journal compilation © 2011 FEBS.

B700, a murine melanoma-specific antigen, binds Vitamin D3; conservation of binding among albuminoid molecules

International Nuclear Information System (INIS)

Farzaneh, N.K.; Walden, T.L. Jr.; Hearing, V.J.; Gersten, D.M.

1990-01-01

B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members of this family include serum albumin (SMA), a-fetoprotein (AFP), vitamin D binding protein (DBP), and C700. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate are largely unknown. The authors examined the functional characteristics of MSA, AFP, and DBP, and for their ability to specifically bind [ 3 H]-1,25-dihydroxy-vitamin D 3 . Scatchard analysis revealed a single binding site for B700 with a Kd of 51,000 M and a Bmax of 4.51 x 10 -7 . There is no significant difference between the Kd and Bmax values among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition of the 1,25-dihydroxy vitamin D 3 with other steroids. 2nM of vitamin D 3 , vitamin D 2 , or estrogen competed for the specific binding of 1,25-dihydroxy vitamin D 3 by B700 but not by DBP. The MSA binding site for 1,25 dihydroxy vitamin D 3 more closely resembles that of DBP than B700. These data indicate that the binding function of the albuminoid proteins has been conserved in the B700 melanoma antigen

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

Science.gov (United States)

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

International Nuclear Information System (INIS)

Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

2008-01-01

The putative ABC transporter ATP-binding protein TM0222 from T. maritima was cloned, overproduced, purified and crystallized. A complete MAD diffraction data set has been collected to 2.3 Å resolution. Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6 4 22, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å 3 Da −1 , which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

Science.gov (United States)

Park, Yoojin; Nim-Anussornkul, Duangrat; Vilaivan, Tirayut; Morii, Takashi; Kim, Byeang Hyean

2018-01-15

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex. Copyright © 2017 Elsevier Ltd. All rights reserved.

The hydrogen bonds between Arg423 and Glu472 and other key residues, Asp443, Ser477, and Pro489, are responsible for the formation and a different positioning of TNP-ATP and ATP within the nucleotide-binding site of Na(+)/K(+)-ATPase

Czech Academy of Sciences Publication Activity Database

Lánský, Zdeněk; Kubala, Martin; Ettrich, Rüdiger; Kutý, Michal; Plášek, J.; Teisinger, Jan; Schoner, W.; Amler, Evžen

2004-01-01

Roč. 43, č. 26 (2004), s. 8303-8311 ISSN 0006-2960 R&D Projects: GA MŠk LN00A141; GA ČR GP206/03/D082; GA ČR GA309/02/1479; GA ČR GD305/03/H148 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100001; CEZ:MSM 111300002 Keywords : sodium pump * ATP-binding site * TNP-ATP Subject RIV: CE - Biochemistry Impact factor: 4.008, year: 2004

Synergistic binding of glucose and aluminium ATP to hexokinase from Saccharomyces cerevisiae.

Science.gov (United States)

Woolfitt, A R; Kellett, G L; Hoggett, J G

1988-08-10

The binding of glucose, AlATP and AlADP to the monomeric and dimeric forms of the native yeast hexokinase PII isoenzyme and to the proteolytically modified SII monomeric form was monitored at pH 6.7 by the concomitant quenching of intrinsic protein fluorescence. No fluorescence changes were observed when free enzyme was mixed with AlATP at concentrations up to 7500 microM. In the presence of saturating concentrations of glucose, the maximal quenching of fluorescence induced by AlATP was between 1.5 and 3.5% depending on species, and the average value of [L]0.5, the concentration of ligand at half-saturation, over all monomeric species was 0.9 +/- 0.4 microM. The presence of saturating concentrations of AlATP diminished [L]0.5 for glucose binding by between 260- and 670-fold for hexokinase PII and SII monomers, respectively (dependent on the ionic strength), and by almost 4000-fold for PII dimer. The data demonstrate extremely strong synergistic interactions in the binding of glucose and AlATP to yeast hexokinase, arising as a consequence of conformational changes in the free enzyme induced by glucose and in enzyme-glucose complex induced by AlATP. The synergistic interactions of glucose and AlATP are related to their kinetic synergism and to the ability of AlATP to act as a powerful inhibitor of the hexokinase reaction.

CD4+ T-cell epitope prediction using antigen processing constraints.

Science.gov (United States)

Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J

2016-05-01

T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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.

Study of the nucleotide binding site of the yeast Schizosaccharomyces pombe plasma membrane H+-ATPase using formycin triphosphate-terbium complex

International Nuclear Information System (INIS)

Ronjat, M.; Lacapere, J.J.; Dufour, J.P.; Dupont, Y.

1987-01-01

The plasma membrane of yeasts contains an H+-ATPase similar to the other cation transport ATPases of eukaryotic organisms. This enzyme has been purified and shows H+ transport in reconstituted vesicles. In the presence of Mg2+, formycin triphosphate (FTP) is hydrolyzed by the H+-ATPase and supports H+ transport. When combined with terbium ion, FTP (Tb-FTP) and ATP (Tb-ATP) are no longer hydrolyzed. Competition between Mg-ATP and Tb-FTP for ATP hydrolysis indicates that terbium-associated nucleotides bind to the catalytic site of the H+-ATPase. The fluorescent properties of the Tb-FTP complex were used to study the active site of the H+-ATPase. Fluorescence of Tb-FTP is greatly enhanced upon binding into the nucleotide site of H+-ATPase with a dissociation constant of 1 microM. Tb-ATP, Tb-ADP, and Tb-ITP are competitive inhibitors of Tb-FTP binding with Ki = 4.5, 5.0, and 6.0 microM, respectively. Binding of Tb-FTP is observed only in the presence of an excess of Tb3+ with an activation constant Ka = 25 microM for Tb3+. Analysis of the data reveals that the sites for Tb-FTP and Tb3+ binding are independent entities. In standard conditions these sites would be occupied by Mg-ATP and Mg2+, respectively. These findings suggest an important regulatory role of divalent cations on the activity of H+-ATPase. Replacement of H 2 O by D 2 O in the medium suggests the existence of two types of nucleotide binding sites differing by the hydration state of the Tb3+ ion in the bound Tb-FTP complex

Protein antigenic structures recognized by T cells: potential applications to vaccine design.

Science.gov (United States)

Berzofsky, J A; Cease, K B; Cornette, J L; Spouge, J L; Margalit, H; Berkower, I J; Good, M F; Miller, L H; DeLisi, C

1987-08-01

In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic

Properties of the manganese(II) binding site in ternary complexes of Mnter dot ADP and Mnter dot ATP with chloroplast coupling factor 1: Magnetic field dependence of solvent sup 1 H and sup 2 H NMR relaxation rates

Energy Technology Data Exchange (ETDEWEB)

Haddy, A.E.; Frasch, W.D.; Sharp, R.R. (Univ. of Michigan, Ann Arbor (USA))

1989-05-02

The influence of the binding of ADP and ATP on the high-affinity Mn(II) binding site of chloroplast coupling factor 1 (CF{sub 1}) was studied by analysis of field-dependent solvent proton and deuteron spin-lattice relaxation data. In order to characterize metal-nucleotide complexes of CF{sub 1} under conditions similar to those of the NMR experiments, the enzyme was analyzed for bound nucleotides and Mn(II) after incubation with AdN and MnCl{sub 2} and removal of labile ligands by extensive gel filtration chromatography. In the field-dependent NMR experiments, the Mn(II) binding site of CF{sub 1} was studied for three mole ratios of added Mn(II) to CF{sub 1}, 0.5, 1.0, and 1.5, in the presence of an excess of either ADP or ATP. The results were extrapolated to zero Mn(II) concentration to characterize the environment of the first Mn(II) binding site of Cf{sub 1}. In the presence of both adenine nucleotides, pronounced changes in the Mn(II) environment relative to that in Mn(II)-CF{sub 1} were evident; the local relaxation rate maxima were more pronounced and shifted to higher field strengths, and the relaxation rate per bound Mn(II) increased at all field strengths. Analysis of the data revealed that the number of exchangeable water molecules liganded to bound Mn(II) increased from one in the binary Mn(II)-CF{sub 1} complex to three and two in the ternary Mn(II)-ADP-CF{sub 1} and Mn(II)-ATP-CF{sub 1} complexes, respectively; these results suggest that a water ligand to bound Mn(II) in the Mn(II)-ADP-CF{sub 1} complex is replaced by the {gamma}-phosphate of ATP in the Mn(II)-ATP-CF{sub 1} complex. A binding model is presented to account for these observations.

Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

International Nuclear Information System (INIS)

Pougeois, R.; Lauquin, G.J.

1985-01-01

The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (P /sub i/ ), could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca 2+ -ATPase in the presence of Ca 2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase, abolished P /sub i/ binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites

A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

DEFF Research Database (Denmark)

Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

2015-01-01

Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells ofte...... or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/....

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

Science.gov (United States)

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

Interaction of ATP with a small heat shock protein from Mycobacterium leprae: effect on its structure and function.

Science.gov (United States)

Nandi, Sandip Kumar; Chakraborty, Ayon; Panda, Alok Kumar; Ray, Sougata Sinha; Kar, Rajiv Kumar; Bhunia, Anirban; Biswas, Ashis

2015-03-01

Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.

Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

Directory of Open Access Journals (Sweden)

Adrien Nicolaï

Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

Analysis of Swine Leukocyte Antigen Peptide Binding Profiles and the Identification of T cell Epitopes by Tetramer Staining

DEFF Research Database (Denmark)

Pedersen, Lasse Eggers

class I peptide binding characteristics in relation to immune responses to vaccination or infection. Applying proven technologies to newly produced, recombinant swine leukocyte antigen (SLA) class I proteins yielded a body of data for peptide:SLA:β2m (pSLA) complex affinity and stability. Mapping...... system to specifically identify and react upon non-self peptide fragments unique only to the foreign intruder. The polymorphism of the MHC molecule effectively individualizes the immune response of each member of any given species. Moreover, responding T cells recognize antigen ligands, only...... in the context of peptide:MHC:β2m (pMHC) complex. The gene encoding the MHC is one of the most polymorphic regions of the genome known. Despite thousands of different human leukocyte antigen (HLA) variants identified, each member of a species only inherits and expresses a few of these MHC alleles. The “MHC...

The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor

OpenAIRE

1990-01-01

T cell-specific expression of the human T cell receptor alpha (TCR- alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR- alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP ...

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

OpenAIRE

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

1998-01-01

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...

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

DEFF Research Database (Denmark)

Kelso, A; Owens, T

1988-01-01

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An ass...

Competitor analogs for defined T cell antigens: peptides incorporating a putative binding motif and polyproline or polyglycine spacers.

Science.gov (United States)

Maryanski, J L; Verdini, A S; Weber, P C; Salemme, F R; Corradin, G

1990-01-12

We describe a new approach for modeling antigenic peptides recognized by T cells. Peptide A24 170-182 can compete with other antigenic peptides that are recognized by H-2kd-restricted cytolytic T cells, presumably by binding to the Kd molecule. By comparing substituted A24 peptides as competitors in a functional competition assay, the A24 residues Tyr-171, Thr-178, and Leu-179 were identified as possible contact residues for Kd. A highly active competitor peptide analog was synthesized in which Tyr was separated from the Thr-Leu pair by a pentaproline spacer. The choice of proline allowed the prediction of a probable conformation for the analog when bound to the Kd molecule. The simplest conformation of the A24 peptide that allows the same spacing and orientation of the motif as in the analog would be a nearly extended polypeptide chain incorporating a single 3(10) helical turn or similar structural kink.

L1198F Mutation Resensitizes Crizotinib to ALK by Altering the Conformation of Inhibitor and ATP Binding Sites

Directory of Open Access Journals (Sweden)

Jian Li

2017-02-01

Full Text Available The efficacy of anaplastic lymphoma kinase (ALK positive non-small-cell lung cancer (NSCLC treatment with small molecule inhibitors is greatly challenged by acquired resistance. A recent study reported the newest generation inhibitor resistant mutation L1198F led to the resensitization to crizotinib, which is the first Food and Drug Administration (FDA approved drug for the treatment of ALK-positive NSCLC. It is of great importance to understand how this extremely rare event occurred for the purpose of overcoming the acquired resistance of such inhibitors. In this study, we exploited molecular dynamics (MD simulation to dissect the molecular mechanisms. Our MD results revealed that L1198F mutation of ALK resulted in the conformational change at the inhibitor site and altered the binding affinity of ALK to crizotinib and lorlatinib. L1198F mutation also affected the autoactivation of ALK as supported by the identification of His1124 and Tyr1278 as critical amino acids involved in ATP binding and phosphorylation. Our findings are valuable for designing more specific and potent inhibitors for the treatment of ALK-positive NSCLC and other types of cancer.

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

KAUST Repository

Lee, Seung-Joo

2010-03-28

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

Science.gov (United States)

Heijnen, Ingmar A F M; Barnett, David; Arroz, Maria J; Barry, Simon M; Bonneville, Marc; Brando, Bruno; D'hautcourt, Jean-Luc; Kern, Florian; Tötterman, Thomas H; Marijt, Erik W A; Bossy, David; Preijers, Frank W M B; Rothe, Gregor; Gratama, Jan W

2004-11-01

HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells. (c) 2004 Wiley-Liss, Inc.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

Energy Technology Data Exchange (ETDEWEB)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

2012-03-02

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

Crystal Structure of the Phage T4 Recombinase UvsX and Its Functional Interaction with the T4 SF2 Helicase UvsW

Energy Technology Data Exchange (ETDEWEB)

Gajewski, Stefan; Webb, Michael R.; Galkin, Vitold; Egelman, Edward H.; Kreuzer, Kenneth N.; White, Stephen W. (Duke); (UV); (SJCH)

2012-07-11

Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.

Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

Science.gov (United States)

Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

2014-01-01

The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

A web server for analysis, comparison and prediction of protein ligand binding sites.

Science.gov (United States)

Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

2016-03-25

One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed β₂-Microglobulin through Distinct Binding Sites

DEFF Research Database (Denmark)

Merkle, Patrick S.; Irving, Melita; Hongjian, Song

2017-01-01

from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of β2m on T-cell cytotoxicity, suggesting an alternate role for β2m binding. Overall, we show that binding of β2m to the TCR occurs in vitro and......T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics...... are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR...

Extracellular ATP reduces HIV-1 transfer from immature dendritic cells to CD4+ T lymphocytes

Directory of Open Access Journals (Sweden)

Barat Corinne

2008-03-01

Full Text Available Abstract Background Dendritic cells (DCs are considered as key mediators of the early events in human immunodeficiency virus type 1 (HIV-1 infection at mucosal sites. Previous studies have shown that surface-bound virions and/or internalized viruses found in endocytic vacuoles of DCs are efficiently transferred to CD4+ T cells. Extracellular adenosine triphosphate (ATP either secreted or released from necrotic cells induces a distorted maturation of DCs, transiently increases their endocytic capacity and affects their migratory capacity. Knowing that high extracellular ATP concentrations are present in situations of tissue injury and inflammation, we investigated the effect of ATP on HIV-1 transmission from DCs to CD4+ T lymphocytes. Results In this study, we show that extracellular ATP reduces HIV-1 transfer from immature monocyte-derived DCs (iDCs to autologous CD4+ T cells. This observed decrease in viral replication was related to a lower proportion of infected CD4+ T cells following transfer, and was seen with both X4- and R5-tropic isolates of HIV-1. Extracellular ATP had no effect on direct CD4+ T cell infection as well as on productive HIV-1 infection of iDCs. These observations indicate that extracellular ATP affects HIV-1 infection of CD4+ T cells in trans with no effect on de novo virus production by iDCs. Additional experiments suggest that extracellular ATP might modulate the trafficking pathway of internalized virions within iDCs leading to an increased lysosomal degradation, which could be partly responsible for the decreased HIV-1 transmission. Conclusion These results suggest that extracellular ATP can act as a factor controlling HIV-1 propagation.

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

Science.gov (United States)

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

Energy Technology Data Exchange (ETDEWEB)

Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

International Nuclear Information System (INIS)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

2014-01-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

Science.gov (United States)

Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-01-01

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

In silico and cell-based analyses reveal strong divergence between prediction and observation of T-cell-recognized tumor antigen T-cell epitopes.

Science.gov (United States)

Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel

2017-07-14

Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Autoradiographic localization of GABA-regulated chloride ionophore binding site using t-[3H]butylbicycloorthobenzoate (TBOB)

International Nuclear Information System (INIS)

O'Connor, L.H.; McEwen, B.S.

1986-01-01

t-Butylbicycloorthobenzoate (TBOB) has been shown to bind with high affinity to sites on or near the chloride ionophore in rat brain membrane preparations. The present study used in vitro quantitative autoradiography to localize the regional distribution of [ 3 H]TBOB binding sites in rat forebrain. Receptors were labelled with 10 nM [ 3 H]TBOB. Nonspecific binding was determined by adding 10 μM picrotoxin to the incubation. Autoradiograms were generated using LKB Ultrofilm and then quantitated using computer-assisted spot-densitometry. The highest specific binding was found in frontal cortex layer 4, islands of Calleja, and ventral palladium. High binding was also found in many regions including anterior hypothalamic n., ventromedial hypothalamic n., dentate gyrus, stratum oriens and stratum lacunosum moleculare of hippocampus, and substantia nigra. Nonspecific binding represented 5 to 15% of total binding and was uniformly low throughout all brain regions. Thus, this selective probe for GABA-regulated chloride ionophore binding sites should provide a useful tool for characterizing this system and its relationship to convulsant and depressant drug action

Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

International Nuclear Information System (INIS)

Ahluwalia, Unnati; Deep, Shashank

2012-01-01

Highlights: ► Interaction between ATP and cyt c takes place via coupled binding–folding mechanism. ► Binding of ATP to cyt c is endothermic. ► GTP and CTP induce similar level of helicity in acid-denatured cyt c as with ATP. ► Compactness induced by ATP is far greater than ADP or AMP. - Abstract: The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1 mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25 °C, however, not in the presence of 1 mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

The Role of ATP-Binding Cassette Transporters in Neuro-Inflammation: Relevance for Bioactive Lipids

OpenAIRE

Kooij, Gijs; van Horssen, Jack; Bandaru, Veera Venkata Ratnam; Haughey, Norman J.; de Vries, Helga E.

2012-01-01

ATP-binding cassette (ABC) transporters are highly expressed by brain endothelial cells that form the blood-brain barrier (BBB). These efflux pumps play an important role in maintaining brain homeostasis as they actively hinder the entry of unwanted blood-derived compounds into the central nervous system (CNS). Consequently, their high activity at the BBB has been a major hurdle for the treatment of several brain diseases, as they prevent numerous drugs to reach their site of action within th...

Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

Energy Technology Data Exchange (ETDEWEB)

B Akabayov; C Richardson

2011-12-31

Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

Science.gov (United States)

Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

2013-01-01

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

DEFF Research Database (Denmark)

Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

2015-01-01

Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells often...... target diverse regions in highly variable viral pathogens and this diversity may need to be addressed through redefinition of suitable peptide targets. Methods: We have developed a method for antigen assessment and target selection for polyvalent vaccines, with which we identified immune epitopes from...... variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results: We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased...

Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

International Nuclear Information System (INIS)

Mueller, H.; Fehr, J.

1986-01-01

The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance.

Science.gov (United States)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-07-05

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable.

The spacing between adjacent binding sites in the family of repeats affects the functions of Epstein-Barr nuclear antigen 1 in transcription activation and stable plasmid maintenance

International Nuclear Information System (INIS)

Hebner, Christy; Lasanen, Julie; Battle, Scott; Aiyar, Ashok

2003-01-01

Epstein-Barr virus (EBV) and the closely related Herpesvirus papio (HVP) are stably replicated as episomes in proliferating latently infected cells. Maintenance and partitioning of these viral plasmids requires a viral sequence in cis, termed the family of repeats (FR), that is bound by a viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). Upon binding FR, EBNA1 maintains viral genomes in proliferating cells and activates transcription from viral promoters required for immortalization. FR from either virus encodes multiple binding sites for the viral maintenance protein, EBNA1, with the FR from the prototypic B95-8 strain of EBV containing 20 binding sites, and FR from HVP containing 8 binding sites. In addition to differences in the number of EBNA1-binding sites, adjacent binding sites in the EBV FR are typically separated by 14 base pairs (bp), but are separated by 10 bp in HVP. We tested whether the number of binding sites, as well as the distance between adjacent binding sites, affects the function of EBNA1 in transcription activation or plasmid maintenance. Our results indicate that EBNA1 activates transcription more efficiently when adjacent binding sites are separated by 10 bp, the spacing observed in HVP. In contrast, using two separate assays, we demonstrate that plasmid maintenance is greatly augmented when adjacent EBNA1-binding sites are separated by 14 bp, and therefore, presumably lie on the same face of the DNA double helix. These results provide indication that the functions of EBNA1 in transcription activation and plasmid maintenance are separable

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors

DEFF Research Database (Denmark)

Jamnani, Fatemeh Rahimi; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali

2014-01-01

Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination...

Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

Science.gov (United States)

Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

2000-03-01

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

Isolation and characterization of antigen-Ia complexes involved in T cell recognition

DEFF Research Database (Denmark)

Buus, S; Sette, A; Colon, S M

1986-01-01

Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes, it is he......Using equilibrium dialysis, it has been previously demonstrated that immunogenic peptides bind specifically to the Ia molecules serving as restriction elements in the immune response to these antigens. Using gel filtration to study the formation of ovalbumin (OVA) peptide-I-Ad complexes...... with glutaraldehyde revealed that the ovalbumin peptide was cross-linked solely to the alpha chain of I-Ad. Planar membranes containing I-Ad-OVA complexes stimulated a T cell response with 2 X 10(4) less antigen than required when uncomplexed antigen was used, thus demonstrating the biologic importance...

Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

Energy Technology Data Exchange (ETDEWEB)

Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

1988-07-12

Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg/sup 2 +/ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with (/sup 32/P)P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca/sup 2 +/ as the activating cation.

ATP-binding motifs play key roles in Krp1p, kinesin-related protein 1, function for bi-polar growth control in fission yeast

International Nuclear Information System (INIS)

Rhee, Dong Keun; Cho, Bon A; Kim, Hyong Bai

2005-01-01

Kinesin is a microtubule-based motor protein with various functions related to the cell growth and division. It has been reported that Krp1p, kinesin-related protein 1, which belongs to the kinesin heavy chain superfamily, localizes on microtubules and may play an important role in cytokinesis. However, the function of Krp1p has not been fully elucidated. In this study, we overexpressed an intact form and three different mutant forms of Krp1p in fission yeast constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif (LZiP). We observed hyper-extended microtubules and the aberrant nuclear shape in Krp1p-overexpressed fission yeast. As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered cell polarity. Additionally, truncation of the leucine zipper-like domain (LZiP) at the C-terminal of Krp1p showed a normal nuclear division. Taken together, we suggest that krp1p is involved in regulation of cell-polarized growth through ATP-binding motifs in fission yeast

Critical role of γ-phosphate in structural transition of Na,K-ATPase upon ATP binding

Science.gov (United States)

Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Klimanova, Elizaveta A.; Dergousova, Elena A.; Lopina, Olga D.; Makarov, Alexander A.

2014-06-01

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that β-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while γ-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the ``E1-open'' to ``E1-closed'' conformation ready for phosphorylation.

Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

KAUST Repository

Weng, Jingwei; Gu, Shuo; Gao, Xin; Huang, Xuhui; Wang, Wenning

2017-01-01

Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

KAUST Repository

Weng, Jingwei

2017-02-23

Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

Association of ATP-Binding Cassette Transporter A1 Gene Polymorphisms in Type 2 Diabetes Mellitus among Malaysians

OpenAIRE

Haghvirdizadeh, Polin; Ramachandran, Vasudevan; Etemad, Ali; Heidari, Farzad; Ghodsian, Nooshin; Bin Ismail, Norzian; Ismail, Patimah

2015-01-01

Background. Type 2 diabetes mellitus (T2DM) is a complex polygenic disorder characterized by impaired insulin resistance, insulin secretion, and dysregulation of lipid and protein metabolism with environmental and genetic factors. ATP-binding cassette transporter A1 (ABCA1) gene polymorphisms are reported as the one of the genetic risk factors for T2DM in various populations with conflicting results. This study was conducted based on PCR-HRM to determine the frequency of ABCA1 gene by rs22308...

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

Science.gov (United States)

Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

2017-09-02

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

Supplementary data: Novel mutation in ATP-binding domain of ...

Indian Academy of Sciences (India)

Novel mutation in ATP-binding domain of ABCD1 gene in adrenoleucodystrophy. Neeraj Kumar, Krishna K. Taneja, Atul Kumar, Deepti Nayar, Bhupesh Taneja, Satindra Aneja,. Madhuri Behari, Veena Kalra and Surendra K. Bansal. J. Genet. 89, 473–477. Figure 1. Rmsd plot of native and Arg617Ser substituted models.

Binding of monoclonal antibody to protein antigen in fluid phase or bound to solid supports

Energy Technology Data Exchange (ETDEWEB)

Kennel, S J

1982-01-01

Rat monoclonal antibody (MoAb) to fragment D (FgD) of human fibrinogen was used to characterize the direct binding of antibody to protein in solution or bound to solid supports. Purified IgG, F(ab')/sub 2/ and Fab' were prepared from ascites fluid of hybridoma 104-14B which is a fusion product of spleen cells from a rat immunized with FgD and the mouse myeloma cell line, P3-X63-Ag8. Two-dimensional electrophoresis of radioiodinated antibody preparations demonstrated the presence of hybrid immunoglobulin molecules, but only structures having rat heavy and rat light chains had active antibody combinig sites. The affinity constant for IgG as well as F(ab')/sub 2/ and Fab', 6x10/sup 9/ M/sup -1/, was identical when tested using fluid phase antigen (/sup 125/I-labeled FgD). Affinity constants determined for direct binding of iodinated IgG using FgD immobilized on solid supports showed a slight dependence on the antigen concentration used in the measurement. These values ranged from 0.5x10/sup 9/ M/sup -1/ at high antigen concentrations (1.3x10/sup -7/ M) to 9x10/sup 9/ M/sup -1/ at low antigen concentration (1.3x10/sup -10/ M). Binding constants for F(ab')/sub 2/ and Fab' gave similar results indicating that binding was homogeneous and univalent. The capacity of solid state antigen to bind antibody varied with the method used to bind FgD to the solid support. FgD bound directly to polystyrene plates was least efficient at binding labeled antibody; FgD bound to plates through intermediate carriers poly(L-lysine) was only slightly more efficient, while antigen bound to Sepharose beads by cyanogen bromide activation was the most active.

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

International Nuclear Information System (INIS)

Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi; Takenaka, Hitoshi; Hamada, Minoru; Kuby, S.A.

1990-01-01

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP 2- and for AMP 2- in human cytosolic adenylate kinase, the authors have investigated the enzymic effects of replacement of the arginine residues, which had been assumed by Pai et al. to interact with the phosphoryl groups of AMP 2- and MgATP 2- . With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the K m,app values for AMP 2- of the mutant enzymes, the relatively small increases in the K m,app values for MgATP 2- , and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. have been reversed and that their ATP-binding site may be assigned as the AMP site

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

International Nuclear Information System (INIS)

Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

1988-01-01

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

Directory of Open Access Journals (Sweden)

Marc Cartellieri

2010-01-01

Full Text Available CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs. First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3

International Nuclear Information System (INIS)

Yoshida, M.; Allison, W.S.

1986-01-01

Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1). One class is comprised of three sites which saturate with [ 3 H]ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP. The binding of ADP to these sites is dependent on Mg2+. [ 3 H]ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium. The other class is comprised of a single site which saturates with [ 3 H]ADP in 30 min with a Kd of 30 microM. [ 3 H]ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium. Binding of [ 3 H]ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM. [ 3 H]ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+. Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide. Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan. Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

Science.gov (United States)

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

Science.gov (United States)

Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

2015-07-03

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of ATP in the Regulation of NCAM Function

DEFF Research Database (Denmark)

Hübschmann, Martin; Skladchikova, Galina

2008-01-01

overlaps with the site of NCAM-FGFR interaction, and ATP is capable of disrupting NCAM-FGFR binding. This implies that NCAM signaling through FGFR can be regulated by ATP, which is supported by the observation that ATP can abrogate NCAM-induced neurite outgrowth. Finally, ATP can induce NCAM ectodomain...... shedding, possibly affecting the structural plasticity associated with learning and memory....

ATP-binding cassette (ABC) transporters in normal and pathological lung

NARCIS (Netherlands)

van der Deen, M; de Vries, EGE; Timens, W; Scheper, RJ; Timmer-Bosscha, H; Postma, DS

2005-01-01

ATP-binding cassette ( ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein ( P-gp), multidrug resistance-associated protein 1 ( MRP1) and

Functional analysis of the ATP-binding cassette (ABC) transporter gene family of Tribolium castaneum.

Science.gov (United States)

Broehan, Gunnar; Kroeger, Tobias; Lorenzen, Marcé; Merzendorfer, Hans

2013-01-16

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly.

Science.gov (United States)

Davies, Brian A; Azmi, Ishara F; Payne, Johanna; Shestakova, Anna; Horazdovsky, Bruce F; Babst, Markus; Katzmann, David J

2010-10-01

ESCRT-III undergoes dynamic assembly and disassembly to facilitate membrane exvagination processes including multivesicular body (MVB) formation, enveloped virus budding, and membrane abscission during cytokinesis. The AAA-ATPase Vps4 is required for ESCRT-III disassembly, however the coordination of Vps4 ATP hydrolysis with ESCRT-III binding and disassembly is not understood. Vps4 ATP hydrolysis has been proposed to execute ESCRT-III disassembly as either a stable oligomer or an unstable oligomer whose dissociation drives ESCRT-III disassembly. An in vitro ESCRT-III disassembly assay was developed to analyze Vps4 function during this process. The studies presented here support a model in which Vps4 acts as a stable oligomer during ATP hydrolysis and ESCRT-III disassembly. Moreover, Vps4 oligomer binding to ESCRT-III induces coordination of ATP hydrolysis at the level of individual Vps4 subunits. These results suggest that Vps4 functions as a stable oligomer that acts upon individual ESCRT-III subunits to facilitate ESCRT-III disassembly.

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

Directory of Open Access Journals (Sweden)

Donald R. Shaffer

2014-01-01

Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

Development of an epitope panel for consistent identification of antigen-specific T-cells in humans

DEFF Research Database (Denmark)

Fløe, Andreas; Løppke, Caroline; Hilberg, Ole

2017-01-01

Objective We aimed to establish a panel of MHC-peptide multimers suitable as a positive control in detection of HLA A*0201 restricted antigen specific T-cells (ASTC) by flow cytometry. Materials and methods MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melano...

Characterization of highly frequent epitope-specific CD45RA+/CCR7+/- T lymphocyte responses against p53-binding domains of the human polyomavirus BK large tumor antigen in HLA-A*0201+ BKV-seropositive donors

Directory of Open Access Journals (Sweden)

Zajac Paul

2006-11-01

Full Text Available Abstract Human polyomavirus BK (BKV has been implicated in oncogenic transformation. Its ability to replicate is determined by the binding of its large tumor antigen (LTag to products of tumor-suppressor genes regulating cell cycle, as specifically p53. We investigated CD8+ T immune responses to BKV LTag portions involved in p53 binding in HLA-A*0201+ BKV LTag experienced individuals. Peptides selected from either p53-binding region (LTag351–450 and LTag533–626 by current algorithms and capacity to bind HLA-A*0201 molecule were used to stimulate CD8+ T responses, as assessed by IFN-γ gene expression ex vivo and detected by cytotoxicity assays following in vitro culture. We observed epitope-specific immune responses in all HLA-A*0201+ BKV LTag experienced individuals tested. At least one epitope, LTag579–587; LLLIWFRPV, was naturally processed in non professional antigen presenting cells and induced cytotoxic responses with CTL precursor frequencies in the order of 1/20'000. Antigen specific CD8+ T cells were only detectable in the CD45RA+ subset, in both CCR7+ and CCR7- subpopulations. These data indicate that widespread cellular immune responses against epitopes within BKV LTag-p53 binding regions exist and question their roles in immunosurveillance against tumors possibly associated with BKV infection.

Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

International Nuclear Information System (INIS)

Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin

2014-01-01

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells

Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

Energy Technology Data Exchange (ETDEWEB)

Diaz, Jason; Wang, Xin; Tsang, Sabrina H. [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States); Jiao, Jing [Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104 (United States); You, Jianxin, E-mail: jianyou@mail.med.upenn.edu [Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 (United States)

2014-07-08

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.

Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

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Pengfei Fang

2015-12-01

Full Text Available Aminoacyl-tRNA synthetases (aaRSs are enzymes that catalyze the transfer of amino acids to their cognate tRNAs as building blocks for translation. Each of the aaRS families plays a pivotal role in protein biosynthesis and is indispensable for cell growth and survival. In addition, aaRSs in higher species have evolved important non-translational functions. These translational and non-translational functions of aaRS are attractive for developing antibacterial, antifungal, and antiparasitic agents and for treating other human diseases. The interplay between amino acids, tRNA, ATP, EF-Tu and non-canonical binding partners, had shaped each family with distinct pattern of key sites for regulation, with characters varying among species across the path of evolution. These sporadic variations in the aaRSs offer great opportunity to target these essential enzymes for therapy. Up to this day, growing numbers of aaRS inhibitors have been discovered and developed. Here, we summarize the latest developments and structural studies of aaRS inhibitors, and classify them with distinct binding modes into five categories.

Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

Science.gov (United States)

Cole, David K.; Bulek, Anna M.; Dolton, Garry; Schauenberg, Andrea J.; Szomolay, Barbara; Trimby, Andrew; Jothikumar, Prithiviraj; Fuller, Anna; Skowera, Ania; Rossjohn, Jamie; Zhu, Cheng; Miles, John J.; Wooldridge, Linda; Rizkallah, Pierre J.; Sewell, Andrew K.

2016-01-01

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease. PMID:27183389

Distance between two binding sites of the same antibody molecule

International Nuclear Information System (INIS)

Cser, L.; Gladkikh, I.A.; Ostanevich, Y.M.; Franek, F.; Novotny, J.; Nezlin, R.S.

1978-01-01

Neutron small-angle scattering experiments are reported, aimed at determining the distance between the two binding sites of the same antibody molecule employing complexes of anti-Dnp antibody with an antigenically univalent, high molecular weight ligand. Although the distance values could be determined only with a large statistical error, the data allowed the conclusion that the geometrical parameters of the complexes formed with the early (i.e., precipitating) antibody are significantly different from those of the complexes formed with the late (i.e, non-precipitating) antibody. The data suggest that the precipitating antibody complexed with a high molecular weight antigen assumes an extended shape with an antigen to antigen distance of 35.8 +- 1.3 nm. (Auth.)

Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci

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Salvatore Loguercio

2018-03-01

Full Text Available CCCTC-binding factor (CTCF is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells and developmental stage-specificity (pre-B vs. pro-B in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRβ loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for

ATP Binding cassette transporter gene expression in rat liver progenitor cells

NARCIS (Netherlands)

Ros, J.E.; Roskams, T.A.D.; Geuken, M.; Havinga, R.; Splinter, P.L.; Petersen, B.E.; LaRusso, N.F.; Kolk, van der D.M.; Kuipers, F.; Faber, K.N.; Müller, M.R.; Jansen, P.L.M.

2003-01-01

Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

ATP binding cassette transporter gene expression in rat liver progenitor cells

NARCIS (Netherlands)

Ros, J. E.; Roskams, T. A. D.; Geuken, M.; Havinga, R.; Splinter, P. L.; Petersen, B. E.; LaRusso, N. F.; van der Kolk, D. M.; Kuipers, F.; Faber, K. N.; Müller, M.; Jansen, P. L. M.

2003-01-01

BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

ATP binding cassette transporter gene expression in rat liver progenitor cells

NARCIS (Netherlands)

Ros, J.E.; Roskams, TAD; Geuken, M; Havinga, R; Splinter, PL; Petersen, BE; LaRusso, NF; van der Kolk, D.M.; Kuipers, F; Faber, KN; Muller, M; Jansen, PLM

Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

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Csermely, P; Szamel, M; Resch, K; Somogyi, J

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

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Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N. (NIH); (Kyoto)

2008-07-29

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells.

Science.gov (United States)

Lecciso, Mariangela; Ocadlikova, Darina; Sangaletti, Sabina; Trabanelli, Sara; De Marchi, Elena; Orioli, Elisa; Pegoraro, Anna; Portararo, Paola; Jandus, Camilla; Bontadini, Andrea; Redavid, Annarita; Salvestrini, Valentina; Romero, Pedro; Colombo, Mario P; Di Virgilio, Francesco; Cavo, Michele; Adinolfi, Elena; Curti, Antonio

2017-01-01

Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC) cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML), ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs) with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1), was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1), which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1 + CD39 + DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair

International Nuclear Information System (INIS)

Thiagalingam, S.; Grossman, L.

1991-01-01

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type A motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA

Antigen specific T-cell responses against tumor antigens are controlled by regulatory T cells in patients with prostate cancer.

Science.gov (United States)

Hadaschik, Boris; Su, Yun; Huter, Eva; Ge, Yingzi; Hohenfellner, Markus; Beckhove, Philipp

2012-04-01

Immunotherapy is a promising approach in an effort to control castration resistant prostate cancer. We characterized tumor antigen reactive T cells in patients with prostate cancer and analyzed the suppression of antitumor responses by regulatory T cells. Peripheral blood samples were collected from 57 patients with histologically confirmed prostate cancer, 8 patients with benign prostatic hyperplasia and 16 healthy donors. Peripheral blood mononuclear cells were isolated and antigen specific interferon-γ secretion of isolated T cells was analyzed by enzyme-linked immunospot assay. T cells were functionally characterized and T-cell responses before and after regulatory T-cell depletion were compared. As test tumor antigens, a panel of 11 long synthetic peptides derived from a total of 8 tumor antigens was used, including prostate specific antigen and prostatic acid phosphatase. In patients with prostate cancer we noted a 74.5% effector T-cell response rate compared with only 25% in patients with benign prostatic hyperplasia and 31% in healthy donors. In most patients 2 or 3 tumor antigens were recognized. Comparing various disease stages there was a clear increase in the immune response against prostate specific antigens from intermediate to high risk tumors and castration resistant disease. Regulatory T-cell depletion led to a significant boost in effector T-cell responses against prostate specific antigen and prostatic acid phosphatase. Tumor specific effector T cells were detected in most patients with prostate cancer, especially those with castration resistant prostate cancer. Since effector T-cell responses against prostate specific antigens strongly increased after regulatory T-cell depletion, our results indicate that immunotherapy efficacy could be enhanced by decreasing regulatory T cells. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

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Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

2009-01-01

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 fails 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.

In vitro site selection of a consensus binding site for the Drosophila melanogaster Tbx20 homolog midline.

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Nima Najand

Full Text Available We employed in vitro site selection to identify a consensus binding sequence for the Drosophila melanogaster Tbx20 T-box transcription factor homolog Midline. We purified a bacterially expressed T-box DNA binding domain of Midline, and used it in four rounds of precipitation and polymerase-chain-reaction based amplification. We cloned and sequenced 54 random oligonucleotides selected by Midline. Electromobility shift-assays confirmed that 27 of these could bind the Midline T-box. Sequence alignment of these 27 clones suggests that Midline binds as a monomer to a consensus sequence that contains an AGGTGT core. Thus, the Midline consensus binding site we define in this study is similar to that defined for vertebrate Tbx20, but differs from a previously reported Midline binding sequence derived through site selection.

Milk-induced eczema is associated with the expansion of T cells expressing cutaneous lymphocyte antigen.

Science.gov (United States)

Abernathy-Carver, K J; Sampson, H A; Picker, L J; Leung, D Y

1995-02-01

The extravasation of T cells at sites of inflammation is critically dependent on the activity of homing receptors (HR) involved in endothelial cell recognition and binding. Two such HR (the cutaneous lymphocyte antigen [CLA] and L-selectin) have been shown to be selectively involved in T cell migration to skin and peripheral lymph nodes, respectively. This study was designed to assess the relationship between the organ specificity of an allergic reaction to food and the expression of HR on T cells activated in vitro by the relevant food allergen. Peripheral blood mononuclear cells were isolated from seven milk allergic children with a history of eczema when exposed to milk. All patients had a positive prick skin test and double-blind placebo-controlled food challenge to milk. 10 children with either allergic eosinophilic gastroenteritis or milk-induced enterocolitis and 8 nonatopic adults served as controls. Five-parameter flow cytometry using monoclonal antibodies was used for detection of the specific HR on freshly isolated T cells versus T cell blasts induced by a 6-d incubation with casein, as compared with Candida albicans. After in vitro stimulation with casein, but not C. albicans, patients with milk allergy and atopic dermatitis had a significantly greater percentage of CLA+ T cells (P < 0.01) than controls with milk-induced enterocolitis, allergic eosinophilic gastroenteritis, or nonatopic healthy controls. In contrast, the percentage of L-selectin-expressing T cells did not differ significantly between these groups. These data suggest that after casein stimulation allergic patients with milk-induced skin disease have an expanded population of CLA+ T cells, as compared with nonatopics or allergic patients without skin involvement. We postulate that heterogeneity in the regulation of HR expression on antigen-specific T cells may play a role in determining sites of involvement in tissue-directed allergic responses.

Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

DEFF Research Database (Denmark)

Christensen, C B; Theander, T G

1997-01-01

Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the ......Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20......% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis...

Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes

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Monica Casucci, Attilio Bondanza

2011-01-01

Full Text Available Chimeric antigen receptors (CARs are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb with the signal transduction machinery of the T-cell receptor (TCR. The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT, wherein allorecognition causes both the graft-versus-leukemia (GVL effect and graft-versus-host disease (GVHD. Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

The molecular bases of δ/αβ T cell-mediated antigen recognition.

Science.gov (United States)

Pellicci, Daniel G; Uldrich, Adam P; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B G; de Boer, Renate; Lim, Ricky T; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H M; Gras, Stephanie; Rossjohn, Jamie; Godfrey, Dale I

2014-12-15

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1(+) human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. © 2014 Pellicci et al.

The molecular bases of δ/αβ T cell–mediated antigen recognition

Science.gov (United States)

Pellicci, Daniel G.; Uldrich, Adam P.; Le Nours, Jérôme; Ross, Fiona; Chabrol, Eric; Eckle, Sidonia B.G.; de Boer, Renate; Lim, Ricky T.; McPherson, Kirsty; Besra, Gurdyal; Howell, Amy R.; Moretta, Lorenzo; McCluskey, James; Heemskerk, Mirjam H.M.; Gras, Stephanie

2014-01-01

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1+ human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity. PMID:25452463

A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules

DEFF Research Database (Denmark)

Pandya, Mital; Rasmussen, Michael; Hansen, Andreas

2015-01-01

Major histocompatibility complex (MHC) class I molecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation......, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular...... pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network...

Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters

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David A. Rhodes

2018-04-01

Full Text Available Activation of human Vγ9/Vδ2 T cells by “phosphoantigens” (pAg, the microbial metabolite (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach these problems, we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 T cell receptor (TCR transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules, such as periplakin or RhoB, did not account for the functional variation between HeLa-L and HeLa-M cells. Instead, the data implicate a checkpoint controlling BTN3A1 stability and protein trafficking, acting at an early time point in its maturation. In addition, plasma membrane profiling was used to identify proteins upregulated in HMB-PP-treated HeLa-M. ABCG2, a member of the ATP-binding cassette (ABC transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated

Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters

Science.gov (United States)

Rhodes, David A.; Chen, Hung-Chang; Williamson, James C.; Hill, Alfred; Yuan, Jack; Smith, Sam; Rhodes, Harriet; Trowsdale, John; Lehner, Paul J.; Herrmann, Thomas; Eberl, Matthias

2018-01-01

Activation of human Vγ9/Vδ2 T cells by “phosphoantigens” (pAg), the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach these problems, we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 T cell receptor (TCR) transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules, such as periplakin or RhoB, did not account for the functional variation between HeLa-L and HeLa-M cells. Instead, the data implicate a checkpoint controlling BTN3A1 stability and protein trafficking, acting at an early time point in its maturation. In addition, plasma membrane profiling was used to identify proteins upregulated in HMB-PP-treated HeLa-M. ABCG2, a member of the ATP-binding cassette (ABC) transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated with

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast

DEFF Research Database (Denmark)

Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D.

2014-01-01

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been...

Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

Science.gov (United States)

Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

2013-07-01

Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

A simplified radioimmunoassay for triiodothyronine (T3) using pre-incubated labelled antigen and antibody

International Nuclear Information System (INIS)

Pillai, M.R.A.; Nagvekar, U.H.; Desai, C.N.; Mani, R.S.

1986-01-01

The development of a simplified radioimmunoassay for triiodothyronine (T 3 ) using pre-incubated labelled T 3 and antibody is described. The assay is carried out by adding 50 μl of standard or sample to 0.4 ml of pre-incubated reagent dispensed in assay tubes. The reaction was allowed to proceed for about four hours and the antigen-antibody complex precipitated by the addition of 1 cm 3 of 22% polyethylene glycol solution. Due to the high dissociation constant of T 3 -antibody complex at 37 deg C, the labelled antigen-antibody complex dissociates and thereby the unlabelled antigen binds with the antibody. The sensitivity of this assay is comparable to an assay done by the equilibrium method using the same antibody. Sixty serum samples were analyzed using this method and were compared with the equilibrium assay. (author)

Structural requirements for the interaction between class II MHC molecules and peptide antigens

DEFF Research Database (Denmark)

Sette, A; Buus, S; Appella, E

1990-01-01

of binding, it is possible to define certain structural features of peptides that are associated with the capacity to bind to a particular MHC specificity (IA(d) or IE(d)); 3) IA(d) and IE(d) molecules recognize different and independent structures on the antigen molecule; 4) only about 10% of the single...... IA(d) and IE(d) molecules and their peptide ligands, we found that some structural characteristics apply to both antigen-MHC interactions. In particular, we found: 1) each MHC molecule is capable of binding many unrelated peptides through the same peptide-binding site; 2) despite this permissiveness...... amino acid substitutions tested on two IA(d)- and IE(d)-binding peptides had significant effect on their MHC-binding capacities, while over 80% of these substitutions significantly impaired T cell recognition of the Ia-peptide complex; 5) based on the segregation between residues that are crucial for T...

ATP-induced conformational changes of nucleotide-binding domains in an ABC transporter. Importance of the water-mediated entropic force.

Science.gov (United States)

Hayashi, Tomohiko; Chiba, Shuntaro; Kaneta, Yusuke; Furuta, Tadaomi; Sakurai, Minoru

2014-11-06

ATP binding cassette (ABC) proteins belong to a superfamily of active transporters. Recent experimental and computational studies have shown that binding of ATP to the nucleotide binding domains (NBDs) of ABC proteins drives the dimerization of NBDs, which, in turn, causes large conformational changes within the transmembrane domains (TMDs). To elucidate the active substrate transport mechanism of ABC proteins, it is first necessary to understand how the NBD dimerization is driven by ATP binding. In this study, we selected MalKs (NBDs of a maltose transporter) as a representative NBD and calculated the free-energy change upon dimerization using molecular mechanics calculations combined with a statistical thermodynamic theory of liquids, as well as a method to calculate the translational, rotational, and vibrational entropy change. This combined method is applied to a large number of snapshot structures obtained from molecular dynamics simulations containing explicit water molecules. The results suggest that the NBD dimerization proceeds with a large gain of water entropy when ATP molecules bind to the NBDs. The energetic gain arising from direct NBD-NBD interactions is canceled by the dehydration penalty and the configurational-entropy loss. ATP hydrolysis induces a loss of the shape complementarity between the NBDs, which leads to the dissociation of the dimer, due to a decrease in the water-entropy gain and an increase in the configurational-entropy loss. This interpretation of the NBD dimerization mechanism in concert with ATP, especially focused on the water-mediated entropy force, is potentially applicable to a wide variety of the ABC transporters.

Keragaman Genetik Sekuen Gen ATP Synthase FO Subunit 6 (ATP6 Monyet Hantu (Tarsius Indonesia (GENETIC DIVERSITY STUDY OF ATP6 GENE SEQUENCES OF TARSIERS FROM INDONESIA

Directory of Open Access Journals (Sweden)

Rini Widayanti

2013-07-01

Full Text Available In a conservation effort, the identification of Tarsier species, on the bases of the morphological andmolecular characteristic is necessary. Up to now, the identification of the animals were based on themorphology and vocalizations, which is extremely difficult to identify each, tarsier species. The objective ofthis research was to study the genetic diversity on ATP6 gene of Tarsius sp. Based on sequencing of PCRproduct using primer ATP6F and ATP6R with 681 nts. PCR product. The sequence of ATP6 fragmentswere aligned with other primates from Gene bank with aid of software Clustal W, and were analyzed usingMEGA program version 4.0. Three different nucleotide sites were found (nucleotide no. 288, 321 and 367.The genetic distance based on nucleotide ATP6 sequence calculated using Kimura 2-parameter modelindicated that the smallest genetic distance 0%, biggest 0.8% and average 0, 2%. The phylogenetic treeusing neighbor joining method based on the sequence of nucleotide ATP6 gene could not be used todifferentiate among T. Dianae (from Central Sulawesi, T. Spectrum (from North Sulawesi, T. bancanus(from lampung, South Sumatera and T.bancanus from West Kalimantan.

A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066.

Science.gov (United States)

De Fusco, Claudia; Brear, Paul; Iegre, Jessica; Georgiou, Kathy Hadje; Sore, Hannah F; Hyvönen, Marko; Spring, David R

2017-07-01

Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors. Copyright © 2017. Published by Elsevier Ltd.

ATP Release from Chemotherapy-Treated Dying Leukemia Cells Elicits an Immune Suppressive Effect by Increasing Regulatory T Cells and Tolerogenic Dendritic Cells

Directory of Open Access Journals (Sweden)

Mariangela Lecciso

2017-12-01

Full Text Available Chemotherapy-induced immunogenic cell death can favor dendritic cell (DC cross-priming of tumor-associated antigens for T cell activation thanks to the release of damage-associated molecular patterns, including ATP. Here, we tested the hypothesis that in acute myeloid leukemia (AML, ATP release, along with its well-known immune stimulatory effect, may also contribute to the generation of an immune suppressive microenvironment. In a cohort of AML patients, undergoing combined daunorubicin and cytarabine chemotherapy, a population of T regulatory cells (Tregs with suppressive phenotype, expressing the immune checkpoint programmed cell death protein 1 (PD-1, was significantly increased. Moving from these results, initial in vitro data showed that daunorubicin was more effective than cytarabine in modulating DC function toward Tregs induction and such difference was correlated with the higher capacity of daunorubicin to induce ATP release from treated AML cells. DCs cultured with daunorubicin-treated AML cells upregulated indoleamine 2,3-dioxygenase 1 (IDO1, which induced anti-leukemia Tregs. These data were confirmed in vivo as daunorubicin-treated mice show an increase in extracellular ATP levels with increased number of Tregs, expressing PD-1 and IDO1+CD39+ DCs. Notably, daunorubicin failed to induce Tregs and tolerogenic DCs in mice lacking the ATP receptor P2X7. Our data indicate that ATP release from chemotherapy-treated dying cells contributes to create an immune suppressive microenvironment in AML.

Role of ATP binding and hydrolysis in the gating of the cystic fibrosis transmembrane conductance regulator

Directory of Open Access Journals (Sweden)

Taras Gout

2012-01-01

Full Text Available The CFTR gene is unique within the ATP-binding cassette (ABC protein family, predominantly of transporters, by coding a chloride channel. The gating mechanism of ABC proteins has been characterized by the ATP Switch model in terms cycles of dimer formation and dissociation linked to ATP binding and hydrolysis, respectively. It would be of interest to assess the extent that Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, a functional channel, fits the ATP Switch model for ABC transporters. Additional transporter mechanisms, namely those of Pgp and HlyB, are discussed for perspective. Literature search of databases selected key references in comparing and contrasting the gating mechanism. CFTR is a functional chloride channel facilitating transmembrane anion flow down electrochemical gradients. A dysfunctional CFTR protein results in cystic fibrosis, a fatal pleiotropic disease currently managed symptomatically. Understanding the gating mechanism will help target drug development aimed at alleviating and curing the disease.

Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

Directory of Open Access Journals (Sweden)

Garry Robert F

2011-01-01

Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

International Nuclear Information System (INIS)

Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

1986-01-01

In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

A label-free electrochemiluminescent sensor for ATP detection based on ATP-dependent ligation.

Science.gov (United States)

Zhao, Tingting; Lin, Chunshui; Yao, Qiuhong; Chen, Xi

2016-07-01

In this work, we describe a new label-free, sensitive and highly selective strategy for the electrochemiluminescent (ECL) detection of ATP at the picomolar level via ATP-induced ligation. The molecular-beacon like DNA probes (P12 complex) are self-assembled on a gold electrode. The presence of ATP leads to the ligation of P12 complex which blocks the digestion by Exonuclease III (Exo III). The protected P12 complex causes the intercalation of numerous ECL indicators (Ru(phen)3(2+)) into the duplex DNA grooves, resulting in significantly amplified ECL signal output. Since the ligating site of T4 DNA ligase and the nicking site of Exo III are the same, it involves no long time of incubation for conformation change. The proposed strategy combines the amplification power of enzyme and the inherent high sensitivity of the ECL technique and enables picomolar detection of ATP. The developed strategy also shows high selectivity against ATP analogs, which makes our new label-free and highly sensitive ligation-based method a useful addition to the amplified ATP detection arena. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of actomyosin ATPase activity by troponin-tropomyosin: effect of the binding of the myosin subfragment 1 (S-1) ATP complex

International Nuclear Information System (INIS)

Greene, L.E.; Williams, D.L. Jr.; Eisenberg, E.

1987-01-01

In the authors' model of regulation, the observed lack of cooperativity in the binding of myosin subfragment 1 (S-1) with bound ATP to the troponin-tropomyosin-actin complex (regulated actin) is explained by S-1 ATP having about the same affinity for the conformation of the regulated actin that activates the myosin ATPase activity (turned-on form) and the conformation that does not activate the myosin ATPase activity (turned-off form). This predicts that, in the absence of Ca 2+ , S-1 ATP should not turn on the regulated actin filament. In the present study, they tested this prediction by using either unmodified S-1 or S-1 chemically modified with N,N'-p-phenylenedimaleimide (pPDM S-1) so that functionally it acts like S-1 ATP, although it does not hydrolyze ATP. [ 14 C]pPDM and [ 32 P]ATP were used as tracers. They found that, in the absence of Ca 2+ , neither S-1 ATP nor pPDM S-1 ATP significantly turns on the ATPase activity of the regulated complex of actin and S-1 (acto S-1). In contrast, in the presence of Ca 2+ , pPDM S-1 ATP binding almost completely turns on the regulated acto S-1 ATPase activity. These results can be explained by their original cooperativity model, with pPDM S-1 ATP binding only ≅ 2 fold more strongly to the turned-on form that to the turned-off form of regulated actin. However, the results are not consistent with our alternative model, which predicts that if pPDM S-1 ATP binds to actin in the absence of Ca 2+ but does not turn on the ATPase activity, then it should also turn on the ATPase activity in the presence of Ca 2+

Mechanical Control of ATP Synthase Function: Activation Energy Difference between Tight and Loose Binding Sites

KAUST Repository

Beke-Somfai, Tamás; Lincoln, Per; Nordén, Bengt

2010-01-01

Despite exhaustive chemical and crystal structure studies, the mechanistic details of how FoF1-ATP synthase can convert mechanical energy to chemical, producing ATP, are still not fully understood. On the basis of quantum mechanical calculations

A novel method for radiolabeling antigen-binding receptors of lymphocytes

International Nuclear Information System (INIS)

Choi, Y.S.; Lee, M.S.; Rosenspire, A.J.

1983-01-01

Antigen-binding receptor (ABR) molecules have been selectively radiolabeled and isolated from immunized chicken spleen cells. The specific radiolabeling of the receptors has been accomplished by utilizing a novel technique employing lactoperoxidase (LPO) covalently linked to antigen (Ag) for which human gammaglobulin was used. The cell surface ABRs were first bound to the Ag-LPO conjugates through specific recognition sites on the Ag portion of the conjugates. The bound LPO portions were then allowed to catalyze the radioiodination of the ABRs. After radiolabeling, cells were solubilized with detergents, ABRs still bound to Ag-LPO conjugates were directly isolated from the lysates via immunoaffinity chromatography utilizing an immunoaffinity reagent directed toward the antigen portion of the ABR-Ag-LPO complex. The radioactive materials were then analyzed via SDS-PAGE under reducing conditions. Most of the specifically-labeled and isolated materials were immunoglobulin (Ig). Both the membrane-bound form of the heavy chain as well as the secreted form were detected, along with the light chain. An additional polypeptide was also selectively labeled and isolated along with the Ig. This may be a molecule closely associated with the membrane immunoglobulin on the B-cell surface. (author)

Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites

International Nuclear Information System (INIS)

Doinel, C.; Ropars, C.; Salmon, C.

1978-01-01

Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)

Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers

NARCIS (Netherlands)

Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; van Roermund, Carlo W.; Lopez, Tatiana E.; Dias, Alexandre M. M.; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J.; Trompier, Doriane; Savary, Stéphane

2014-01-01

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned

Specific binding of antigen-antibody in physiological environments: Measurement, force characteristics and analysis

Science.gov (United States)

Gu, Xin; Zhou, Jun; Zhou, Lu; Xie, Shusen; Petti, Lucia; Wang, Shaomin; Wang, Fuyan

2018-05-01

The specific recognition of the antigen by the antibody is the crucial step in immunoassays. Measurement and analysis of the specific recognition, including the ways in which it is influenced by external factors are of paramount significance for the quality of the immunoassays. Using prostate-specific antigen (PSA)/anti-PSA antibody and α-fetoprotein (AFP) /anti-AFP antibody as examples, we have proposed a novel solution for measuring the binding forces between the antigens and their corresponding antibodies in different physiological environments by combining laminar flow control technology and optical tweezers technology. On the basis of the experimental results, the different binding forces of PSA/anti-PSA antibody and AFP/anti-AFP antibody in the same phosphate-buffered saline (PBS) environments are analysed by comparing the affinity constant of the two antibodies and the number of antigenic determinants of the two antigens. In different electrolyte environments, the changes of the binding force of antigens-antibodies are explained by the polyelectrolyte effect and hydrophobic interaction. Furthermore, in different pH environments, the changes of binding forces of antigens-antibodies are attributed to the role of the denaturation of protein. The study aims to recognise the antigen-antibody immune mechanism, thus ensuring further understanding of the biological functions of tumour markers, and it promises to be very useful for the clinical diagnosis of early-stage cancer.

Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme

International Nuclear Information System (INIS)

Zhang Yun-Xin

2014-01-01

Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical mechanics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provide a new way to understand biophysical processe by statistical mechanics analysis. (interdisciplinary physics and related areas of science and technology)

Prediction of FAD binding sites in electron transport proteins according to efficient radial basis function networks and significant amino acid pairs.

Science.gov (United States)

Le, Nguyen-Quoc-Khanh; Ou, Yu-Yen

2016-07-30

Cellular respiration is a catabolic pathway for producing adenosine triphosphate (ATP) and is the most efficient process through which cells harvest energy from consumed food. When cells undergo cellular respiration, they require a pathway to keep and transfer electrons (i.e., the electron transport chain). Due to oxidation-reduction reactions, the electron transport chain produces a transmembrane proton electrochemical gradient. In case protons flow back through this membrane, this mechanical energy is converted into chemical energy by ATP synthase. The convert process is involved in producing ATP which provides energy in a lot of cellular processes. In the electron transport chain process, flavin adenine dinucleotide (FAD) is one of the most vital molecules for carrying and transferring electrons. Therefore, predicting FAD binding sites in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. We used an independent data set to evaluate the performance of the proposed method, which had an accuracy of 69.84 %. We compared the performance of the proposed method in analyzing two newly discovered electron transport protein sequences with that of the general FAD binding predictor presented by Mishra and Raghava and determined that the accuracy of the proposed method improved by 9-45 % and its Matthew's correlation coefficient was 0.14-0.5. Furthermore, the proposed method enabled reducing the number of false positives significantly and can provide useful information for biologists. We developed a method that is based on PSSM profiles and SAAPs for identifying FAD binding sites in newly discovered electron transport protein sequences. This approach achieved a significant improvement after we added SAAPs to PSSM features to analyze FAD binding proteins in the electron transport chain. The proposed method can serve as an effective tool for predicting FAD binding sites in electron

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation.

Science.gov (United States)

Chen, Kai; Duan, Wenxiu; Han, Qianqian; Sun, Xuan; Li, Wenqian; Hu, Shuangyun; Wan, Jiajia; Wu, Jiang; Ge, Yushu; Liu, Dan

2018-03-08

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

ATP-modulated K+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release.

OpenAIRE

Bernardi, H; De Weille, J R; Epelbaum, J; Mourre, C; Amoroso, S; Slama, A; Fosset, M; Lazdunski, M

1993-01-01

The adenohypophysis contains high-affinity binding sites for antidiabetic sulfonylureas that are specific blockers of ATP-sensitive K+ channels. The binding protein has a M(r) of 145,000 +/- 5000. The presence of ATP-sensitive K+ channels (26 pS) has been demonstrated by electrophysiological techniques. Intracellular perfusion of adenohypophysis cells with an ATP-free medium to activate ATP-sensitive K+ channels induces a large hyperpolarization (approximately 30 mV) that is antagonized by an...

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8+ T Cells

Science.gov (United States)

Maji, Mithun; Mazumder, Saumyabrata; Bhattacharya, Souparno; Choudhury, Somsubhra Thakur; Sabur, Abdus; Shadab, Md.; Bhattacharya, Pradyot; Ali, Nahid

2016-06-01

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8+ cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4+ and CD8+ T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4+ and CD8+ T cells. Furthermore, lymphoid CD8+ T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8+ T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8+ T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

Ets transcription factor GABP controls T cell homeostasis and immunity.

Science.gov (United States)

Luo, Chong T; Osmanbeyoglu, Hatice U; Do, Mytrang H; Bivona, Michael R; Toure, Ahmed; Kang, Davina; Xie, Yuchen; Leslie, Christina S; Li, Ming O

2017-10-20

Peripheral T cells are maintained in the absence of vigorous stimuli, and respond to antigenic stimulation by initiating cell cycle progression and functional differentiation. Here we show that depletion of the Ets family transcription factor GA-binding protein (GABP) in T cells impairs T-cell homeostasis. In addition, GABP is critically required for antigen-stimulated T-cell responses in vitro and in vivo. Transcriptome and genome-wide GABP-binding site analyses identify GABP direct targets encoding proteins involved in cellular redox balance and DNA replication, including the Mcm replicative helicases. These findings show that GABP has a nonredundant role in the control of T-cell homeostasis and immunity.

Demonstration of specific binding sites for 3H-RRR-alpha-tocopherol on human erythrocytes

International Nuclear Information System (INIS)

Kitabchi, A.E.; Wimalasena, J.

1982-01-01

Previous work from our laboratory demonstrated specific binding sites for 3 H-RRR-alpha-tocopherol ( 3 H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for 3 H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for 3 H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane

Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

Energy Technology Data Exchange (ETDEWEB)

Xu, Rui; McBride, Ryan; Paulson, James C.; Basler, Christopher F.; Wilson, Ian A. (Sinai); (Scripps)

2010-03-04

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which represent the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

NARCIS (Netherlands)

Rijpma, S.R.; Velden, M. van der; Gonzalez-Pons, M.; Annoura, T.; Schaijk, B.C.L. van; Gemert, G.J.A. van; Heuvel, J.M.W. van den; Ramesar, J.; Chevalley-Maurel, S.; Ploemen, I.H.; Khan, S.M.; Franetich, J.F.; Mazier, D.; Wilt, J.H.W. de; Serrano, A.E.; Russel, F.G.; Janse, C.J.; Sauerwein, R.W.; Koenderink, J.B.; Franke-Fayard, B.M.

2016-01-01

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC

Metabolism of ATP-binding cassette drug transporter inhibitors: complicating factor for multidrug resistance.

NARCIS (Netherlands)

Cnubben, N.H.; Wortelboer, H.M.; Zanden, J.J. van; Rietjens, I.M.; Bladeren, P.J. van

2005-01-01

Membrane transport proteins belonging to the ATP-binding cassette (ABC) family of transport proteins play a central role in the defence of organisms against toxic compounds, including anticancer drugs. However, for compounds that are designed to display a toxic effect, this defence system diminishes

Molecular structure of human KATP in complex with ATP and ADP.

Science.gov (United States)

Lee, Kenneth Pak Kin; Chen, Jue; MacKinnon, Roderick

2017-12-29

In many excitable cells, KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K + channel Kir6.2, in the presence of Mg 2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg 2+ -ATP in the degenerate site and Mg 2+ -ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP's ability to override ATP inhibition. © 2017, Lee et al.

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

Science.gov (United States)

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

Molecular Events Involved in a Single Cycle of Ligand Transfer from an ATP Binding Cassette Transporter, LolCDE, to a Molecular Chaperone, LolA*

OpenAIRE

Taniguchi, Naohiro; Tokuda, Hajime

2008-01-01

An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters med...

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

Science.gov (United States)

Carpentier, Mathieu; Allain, Fabrice; Slomianny, Marie-Christine; Durieux, Sandrine; Vanpouille, Christophe; Haendler, Bernard; Spik, Geneviève

2002-04-23

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

DEFF Research Database (Denmark)

Paulsen, Marianne; Lund, Connie; Akram, Zarqa

2006-01-01

Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional...... effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally...... degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A...

The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

Directory of Open Access Journals (Sweden)

Luciana Galetto

Full Text Available Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP strain, and three others as non-vectors. Interactions between a labelled (recombinant CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

International Nuclear Information System (INIS)

Noda, T.; Satake, M.; Robins, T.; Ito, Y.

1986-01-01

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10 6 cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture

Presentation of lipid antigens to T cells.

Science.gov (United States)

Mori, Lucia; De Libero, Gennaro

2008-04-15

T cells specific for lipid antigens participate in regulation of the immune response during infections, tumor immunosurveillance, allergy and autoimmune diseases. T cells recognize lipid antigens as complexes formed with CD1 antigen-presenting molecules, thus resembling recognition of MHC-peptide complexes. The biophysical properties of lipids impose unique mechanisms for their delivery, internalization into antigen-presenting cells, membrane trafficking, processing, and loading of CD1 molecules. Each of these steps is controlled at molecular and celular levels and determines lipid immunogenicity. Lipid antigens may derive from microbes and from the cellular metabolism, thus allowing the immune system to survey a large repertoire of immunogenic molecules. Recognition of lipid antigens facilitates the detection of infectious agents and the initiation of responses involved in immunoregulation and autoimmunity. This review focuses on the presentation mechanisms and specific recognition of self and bacterial lipid antigens and discusses the important open issues.

Complementation of a primer binding site-impaired murine leukemia virus-derived retroviral vector by a genetically engineered tRNA-like primer

DEFF Research Database (Denmark)

Lund, Anders Henrik; Duch, M; Lovmand, J

1997-01-01

, but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer...... binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology....

Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain.

Science.gov (United States)

Ahn, Jinhi; Beharry, Seelochan; Molday, Laurie L; Molday, Robert S

2003-10-10

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

OpenAIRE

Bolton, Michael J; Garry, Robert F

2011-01-01

Abstract Background The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. Results Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, ...

Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

Science.gov (United States)

Truan, Daphné; Bjelić, Saša; Li, Xiao-Dan; Winkler, Fritz K

2014-07-29

The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Distribution of 125I-monoclonal antibodies to antigen Ly 2.1 of T-lymphocytes in mice under the influence of immunomodulators

International Nuclear Information System (INIS)

Mirolyubova, Sh.Yu.; Fadeev, N.P.; Serzhanina, V.A.; Klimovich, V.B.; Makarenko, M.V.; Korsakova, L.N.

1991-01-01

A study was made of the distribution of 125 I (a chloramine method of labelling) monoclonal antibodies to the surface antigen Ly 2.1 T-lymphocytes during action of immunomodulators (tactivin, hydrocortisone, tactivin administered after hydrocortisone) on ACR mice. These antibodies were shown to retain antigen binding capacity, permitting monitoring of the redistribution of the antigen in the body exposed to immunomodulators

Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

International Nuclear Information System (INIS)

Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

1983-01-01

A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

A radioimmunoassay to screen for antibodies to native conformational antigens and analyse ligand-induced structural states of antigenic proteins

International Nuclear Information System (INIS)

Bernotat-Danielowski, S.; Koepsell, H.

1988-01-01

A radioimmunoassay is described in which antigenic protein was immobilized by incubating nitrocellulose filters of defined diameter with antigen-containing solutions. Antigenic sites which are sensitive to protein denaturation by drying could be detected with the assay. The assay was also used to screen hybridoma supernatants for antibodies directed against Na + cotransport proteins from renal brush-border membranes. Monoclonal antibodies were selected which showed different binding charactertics depending on whether or not substrates of Na + cotransporters were present. One of the antibodies, which showed different antibody binding after addition of D-glucose or L-lactate, bound to a polypeptide component of the renal N + -D-glucose cotransporter and was able to inhibit Na + gradient-dependent. To investigate the effects of D-glucose and L-lactate on the binding of this antibody concentration dependence was measured. High and low affinity binding sites for D-glucose and L-lactate were characterized thereby demonstrating that the radioimmunoassay permits investigations of the properties of high and low affinity substrate binding sites. (author). refs.; 6 figs.; 2 tabs

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

Energy Technology Data Exchange (ETDEWEB)

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface in which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.

Antigen presentation by hapten-specific B lymphocytes. II. Specificity and properties of antigen-presenting B lymphocytes, and function of immunoglobulin receptors

International Nuclear Information System (INIS)

Abbas, A.K.; Haber, S.; Rock, K.L.

1985-01-01

Studies were designed to examine the ability of hapten-binding murine B lymphocytes to present hapten-protein conjugates to protein antigen-specific, Ia-restricted T cell hybridomas. BALB/c B cells specific for TNP or FITC presented hapten-modified proteins (TNP-G1 phi, TNP-OVA, or FITC-OVA) to the relevant T cell hybridomas at concentrations below 0.1 microgram/ml. Effective presentation of the same antigens by B lymphocyte-depleted splenocytes, and of unmodified proteins by either hapten-binding B cells or Ig spleen cells, required about 10(3)-to 10(4)-fold higher concentrations of antigen. The use of two different haptens and two carrier proteins showed that this extremely efficient presentation of antigen was highly specific, with hapten specificity being a property of the B cells and carrier specificity of the responding T cells. The presentation of hapten-proteins by hapten-binding B lymphocytes was radiosensitive and was not affected by the depletion of plastic-adherent cells, suggesting that conventional APCs (macrophages or dendritic cells) are not required in this phenomenon. Antigen-pulsing and antibody-blocking experiments showed that this hapten-specific antigen presentation required initial binding of antigen to surface Ig receptors. Moreover, linked recognition of hapten and carrier determinants was required, but these recognition events could be temporally separated. Finally, an antigen-processing step was found to be necessary, and this step was disrupted by ionizing radiation. These data suggest a role for B cell surface Ig in providing a specific high-affinity receptor to allow efficient uptake or focusing of antigen for its subsequent processing and presentation to T lymphocytes

The Role of ATP-Binding Cassette Transporters in Neuro-Inflammation: Relevance for Bioactive Lipids

NARCIS (Netherlands)

Kooij, G.; van Horssen, J.; Bandaru, V.V.R.; Haughey, N.J.; de Vries, H.E.

2012-01-01

ATP-binding cassette (ABC) transporters are highly expressed by brain endothelial cells that form the blood-brain barrier (BBB). These efflux pumps play an important role in maintaining brain homeostasis as they actively hinder the entry of unwanted blood-derived compounds into the central nervous

Participation of L3T4 in T cell activation in the absence of class II major histocompatibility complex antigens. Inhibition by anti-L3T4 antibodies is a function both of epitope density and mode of presentation of anti-receptor antibody

DEFF Research Database (Denmark)

Owens, T; Fazekas de St Groth, B

1987-01-01

two monoclonal antibodies, KJ16-133.18 and F23.1, that recognize a determinant encoded by the T cell receptor V beta 8 gene family. These antibodies were used to select two clones of T cells with surface phenotype Thy-1.2+, L3T4+, Lyt-2-, KJ16-133.18+, F23.1+, IA-, IE-. One of these clones (E9.D4......The recognition of many class II major histocompatibility complex (MHC)-associated antigens by T cells requires the participation of the L3T4 molecule. It has been proposed that this molecule acts to stabilize low affinity binding to antigen in association with MHC and thereby increases the avidity...... of T cell/antigen interactions. By using antibodies against the T cell antigen receptor (TCR) to activate T cells, thereby circumventing the requirement for antigen presenting cells and MHC-associated antigen, we have been able to study the function of L3T4 in the absence of class II MHC. We have used...

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

Science.gov (United States)

Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

2012-02-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

Science.gov (United States)

Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

2018-02-01

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

International Nuclear Information System (INIS)

Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

2009-01-01

Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme.

Science.gov (United States)

Wieczorek, Anna; Downey, Christopher D; Dallmann, H Garry; McHenry, Charles S

2010-09-17

The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β(2). DNA synthesis activity can be restored by increased concentrations of β(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

Directory of Open Access Journals (Sweden)

Wei Sheng Chia

Full Text Available p97/Valosin-containing protein (VCP is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD. It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

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Chia, Wei Sheng; Chia, Diana Xueqi; Rao, Feng; Bar Nun, Shoshana; Geifman Shochat, Susana

2012-01-01

p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

International Nuclear Information System (INIS)

Alderson, R.; Pastan, I.; Cheng, S.

1985-01-01

The binding of [ 125 I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[ 125 I]T3 (BrAc[ 125 I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (K /sub d/ = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (K /sub k/ = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [ 125 I]T3. The affinity labeling ligand BrAc[ 125 I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[ 125 I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000

On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

International Nuclear Information System (INIS)

Du, Z.; Boyer, P.D.

1990-01-01

Washed chloroplast thylakoid membranes upon exposure to [ 3 H]ADP retain in tightly bound [ 3 H]ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg 2+ results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound [ 3 H]ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg 2+ - and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22 degree C and of about 15 s at 37 degree C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg 2+ and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound [ 3 H]ADP parallels the onset of ATPase activity, although some [ 3 H]ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound [ 3 H]ADP being at a catalytic site and being replaced as this Mg 2+ - and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P i binding site of the enzyme-ADP-Mg 2+ complex to give a form more readily activated by ATP binding at an alternative site

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

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Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

2014-02-01

T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

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Pascal, John M; Armen, Roger S

2012-01-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

Estradiol-induced vaginal mucus inhibits antigen penetration and CD8(+) T cell priming in response to intravaginal immunization.

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Seavey, Matthew M; Mosmann, Tim R

2009-04-14

Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8(+) T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APCs) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8(+) T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8(+) T cell priming after insemination or vaginal vaccination.

Estradiol-induced vaginal mucus inhibits antigen penetration and CD8+ T cell priming in response to intravaginal immunization

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Seavey, Matthew M.; Mosmann, Tim R.

2010-01-01

Although vaginal immunization has been explored as a strategy to induce mucosal immunity in the female reproductive tract, this site displays unique immunological features that probably evolved to inhibit anti-paternal T cell responses after insemination to allow successful pregnancy. We previously demonstrated that estradiol, which induces an estrus-like state, prevented CD8+ T cell priming during intravaginal immunization of mice. We now show that estradiol prevented antigen loading of vaginal antigen presenting cells (APC) after intravaginal immunization. Histological examination confirmed that estradiol prevented penetration of peptide antigen into the vaginal wall. Removal of the estradiol-induced mucus barrier by mucinase partially restored antigen loading of vaginal APC and CD8+ T cell proliferation in vivo. The estradiol-induced mucus barrier may thus prevent exposure to antigens delivered intravaginally, supplementing additional estradiol-dependent mechanism(s) that inhibit CD8+ T cell priming after insemination or vaginal vaccination. PMID:19428849

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

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Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2

Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases

International Nuclear Information System (INIS)

Falo, L.D. Jr.; Haber, S.I.; Herrmann, S.; Benacerraf, B.; Rock, K.L.

1987-01-01

To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) the authors analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A 2 and phospholipase C. In addition it is observed with three distinct antigens - ovalbumin, bovine insulin, and poly(LGlu 56 LLys 35 LPhe 9 )[(GluLysPhe)/sub n/]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to control in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or processing independent antigens. In parallel studies 125 I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125 I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. These studies demonstrate that phospholipase effectively removes processed cell surface antigen

Two separable functional domains of simian virus 40 large T antigen: carboxyl-terminal region of simian virus 40 large T antigen is required for efficient capsid protein synthesis.

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Tornow, J; Polvino-Bodnar, M; Santangelo, G; Cole, C N

1985-01-01

The carboxyl-terminal portion of simian virus 40 large T antigen is essential for productive infection of CV-1 and CV-1p green monkey kidney cells. Mutant dlA2459, lacking 14 base pairs at 0.193 map units, was positive for viral DNA replication, but unable to form plaques in CV-1p cells (J. Tornow and C.N. Cole, J. Virol. 47:487-494, 1983). In this report, the defect of dlA2459 is further defined. Simian virus 40 late mRNAs were transcribed, polyadenylated, spliced, and transported in dlA2459-infected cells, but the level of capsid proteins produced in infected CV-1 green monkey kidney cells was extremely low. dlA2459 large T antigen lacks those residues known to be required for adenovirus helper function, and the block to productive infection by dlA2459 occurs at the same stage of infection as the block to productive adenovirus infection of CV-1 cells. These results suggest that the adenovirus helper function is required for productive infection by simian virus 40. Mutant dlA2459 was able to grow on the Vero and BSC-1 lines of African green monkey kidney cells. Additional mutants affecting the carboxyl-terminal portion of large T were prepared. Mutant inv2408 contains an inversion of the DNA between the BamHI and BclI sites (0.144 to 0.189 map units). This inversion causes transposition of the carboxyl-terminal 26 amino acids of large T antigen and the carboxyl-terminal 18 amino acids of VP1. This mutant was viable, even though the essential information absent from dlA2459 large T antigen has been transferred to the carboxyl terminus of VP1 of inv2408. The VP1 polypeptide carrying this carboxyl-terminal portion of large T could overcome the defect of dlA2459. This indicates that the carboxyl terminus of large T antigen is a separate and separable functional domain. Images PMID:2982029

MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

International Nuclear Information System (INIS)

Milgrom, Elena M.; Milgrom, Yakov M.

2012-01-01

Highlights: ► MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. ► V-ATPase activity saturation with MgATP is not sufficient for complete protection. ► The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K m values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

A new type of Na(+-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif.

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Sarah Schulz

Full Text Available The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F₁F₀-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c₁₁ ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

Cooperative binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate to aspartate transcarbamoylase and the heterotropic effects of ATP and CTP

International Nuclear Information System (INIS)

Newell, J.O.; Markby, D.W.; Schachman, H.K.

1989-01-01

Most investigations of the allosteric properties of the regulatory enzyme aspartate transcarbamoylase (ATCase) from Escherichia coli are based on the sigmoidal dependence of enzyme activity on substrate concentration and the effects of the inhibitor, CTP, and the activator, ATP, on the saturation curves. Interpretations of these effects in terms of molecular models are complicated by the inability to distinguish between changes in substrate binding and catalytic turnover accompanying the allosteric transition. In an effort to eliminate this ambiguity, the binding of the 3H-labeled bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) to aspartate transcarbamoylase in the absence and presence of the allosteric effectors ATP and CTP has been measured directly by equilibrium dialysis at pH 7 in phosphate buffer. PALA binds with marked cooperativity to the holoenzyme with an average dissociation constant of 110 nM. ATP and CTP alter both the average affinity of ATCase for PALA and the degree of cooperativity in the binding process in a manner analogous to their effects on the kinetic properties of the enzyme; the average dissociation constant of PALA decreases to 65 nM in the presence of ATP and increases to 266 nM in the presence of CTP while the Hill coefficient, which is 1.95 in the absence of effectors, becomes 1.35 and 2.27 in the presence of ATP and CTP, respectively. The dissociation constant of PALA from the catalytic subunit is 95 nM. Interpretation of these results in terms of a thermodynamic scheme linking PALA binding to the assembly of ATCase from catalytic and regulatory subunits demonstrates that saturation of the enzyme with PALA shifts the equilibrium between holoenzyme and subunits slightly toward dissociation

Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

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Sumi, Daigo; Asao, Masashi; Okada, Hideta; Yogi, Kuniko; Miyataka, Hideki; Himeno, Seiichiro

2016-06-01

Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity.

[3]tetrahydrotrazodone binding. Association with serotonin binding sites

International Nuclear Information System (INIS)

Kendall, D.A.; Taylor, D.P.; Enna, S.J.

1983-01-01

High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

Role of the Ectodomain Serine 275 in Shaping the Binding Pocket of the ATP-Gated P2X3 Receptor

Czech Academy of Sciences Publication Activity Database

Petrenko, N.; Khafizov, K.; Tvrdoňová, Vendula; Skorinkin, A.; Giniatullin, R.

2011-01-01

Roč. 50, č. 39 (2011), s. 8427-8436 ISSN 0006-2960 Grant - others:Univerzita Karlova(CZ) 3446/2011 Institutional research plan: CEZ:AV0Z50110509 Keywords : P2X3 * purinergic * ATP * ATP-binding pocket * receptor Subject RIV: ED - Physiology Impact factor: 3.422, year: 2011

Crystallization and preliminary X-ray diffraction analysis of an archaeal tRNA-modification enzyme, TiaS, complexed with tRNAIle2 and ATP

International Nuclear Information System (INIS)

Osawa, Takuo; Inanaga, Hideko; Kimura, Satoshi; Terasaka, Naohiro; Suzuki, Tsutomu; Numata, Tomoyuki

2011-01-01

A. fulgidus TiaS was cocrystallized with tRNA Ile2 and ATP and X-ray diffraction data were collected to 2.9 Å resolution using a synchrotron-radiation source. The cytidine at the first anticodon position of archaeal tRNA Ile2 , which decodes the isoleucine AUA codon, is modified to 2-agmatinylcytidine (agm 2 C) to guarantee the fidelity of protein biosynthesis. This post-transcriptional modification is catalyzed by tRNA Ile -agm 2 C synthetase (TiaS) using ATP and agmatine as substrates. Archaeoglobus fulgidus TiaS was overexpressed in Escherichia coli cells and purified. tRNA Ile2 was prepared by in vitro transcription with T7 RNA polymerase. TiaS was cocrystallized with both tRNA Ile2 and ATP by the vapour-diffusion method. The crystals of the TiaS–tRNA Ile2 –ATP complex diffracted to 2.9 Å resolution using synchrotron radiation at the Photon Factory. The crystals belonged to the primitive hexagonal space group P3 2 21, with unit-cell parameters a = b = 131.1, c = 86.6 Å. The asymmetric unit is expected to contain one TiaS–tRNA Ile2 –ATP complex, with a Matthews coefficient of 2.8 Å 3 Da −1 and a solvent content of 61%

In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

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Janet L Smith

2015-05-01

Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

The location and nature of general anesthetic binding sites on the active conformation of firefly luciferase; a time resolved photolabeling study.

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Sivananthaperumal Shanmugasundararaj

Full Text Available Firefly luciferase is one of the few soluble proteins that is acted upon by a wide variety of general anesthetics and alcohols; they inhibit the ATP-driven production of light. We have used time-resolved photolabeling to locate the binding sites of alcohols during the initial light output, some 200 ms after adding ATP. The photolabel 3-azioctanol inhibited the initial light output with an IC50 of 200 µM, close to its general anesthetic potency. Photoincorporation of [(3H]3-azioctanol into luciferase was saturable but weak. It was enhanced 200 ms after adding ATP but was negligible minutes later. Sequencing of tryptic digests by HPLC-MSMS revealed a similar conformation-dependence for photoincorporation of 3-azioctanol into Glu-313, a residue that lines the bottom of a deep cleft (vestibule whose outer end binds luciferin. An aromatic diazirine analog of benzyl alcohol with broader side chain reactivity reported two sites. First, it photolabeled two residues in the vestibule, Ser-286 and Ile-288, both of which are implicated with Glu-313 in the conformation change accompanying activation. Second, it photolabeled two residues that contact luciferin, Ser-316 and Ser-349. Thus, time resolved photolabeling supports two mechanisms of action. First, an allosteric one, in which anesthetics bind in the vestibule displacing water molecules that are thought to be involved in light output. Second, a competitive one, in which anesthetics bind isosterically with luciferin. This work provides structural evidence that supports the competitive and allosteric actions previously characterized by kinetic studies.

Genes regulation encoding ADP/ATP carrier in yeasts Saccharomyces cerevisiae and Candida parapsilosis

International Nuclear Information System (INIS)

Nebohacova, M.

2000-01-01

Genes encoding a mitochondrial ADP/ATP carrier (AAC) in yeast Saccharomyces cerevisiae and Candida parapsilosis were investigated. AAC2 is coding for the major AAC isoform in S. cerevisiae. We suggest that AAC2 is a member of a syn-expression group of genes encoding oxidative phosphorylation proteins. Within our previous studies on the regulation of the AAC2 transcription an UAS (-393/-268) was identified that is essential for the expression of this gene. Two functional regulatory cis-elements are located within this UAS -binding sites for an ABFl factor and for HAP2/3/4/5 heteromeric complex. We examined relative contributions and mutual interactions of the ABFl and HAP2/3/4/5 factors in the activation of transcription from the UAS of the AAC2 gene. The whole UAS was dissected into smaller sub-fragments and tested for (i) the ability to form DNA-protein complexes with cellular proteins in vitro, (ii) the ability to confer heterologous expression using AAC3 gene lacking its own promoter, and (iii) the expression of AAC3-lacZ fusion instead of intact AAC3 gene. The obtained results demonstrated that: a) The whole UAS as well as sub-fragment containing only ABF1-binding site are able to form DNA-protein complexes with cellular proteins in oxygen- and heme- dependent manner. The experiments with antibody against the ABF1 showed that the ABF1 factor is one of the proteins binding to AAC2 promoter. We have been unsuccessful to prove the binding of cellular proteins to the HAP2/3/4/5-binding site. However, the presence of HAP2/3/4/5-binding site is necessary to drive a binding of cellular proteins to the ABF1-binding site in carbon source-dependent manner. b) The presence of both ABF1- and HAP2/3/4/5-binding sites and original spacing between them is necessary to confer the growth of Aaac2 mutant strain on non- fermentable carbon source when put in front of AAC3 gene introduced on centromeric vector to Aaac2 mutant strain. c) For the activation of AAC3-lacZ expression on

Can resting B cells present antigen to T cells

International Nuclear Information System (INIS)

Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

1985-01-01

Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies the authors have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [ 3 H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells

Membrane porters of ATP-binding cassette transport systems are polyphyletic.

Science.gov (United States)

Wang, Bin; Dukarevich, Maxim; Sun, Eric I; Yen, Ming Ren; Saier, Milton H

2009-09-01

The ATP-binding cassette (ABC) superfamily consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP hydrolyzing constituents, which are monophyletic. The evolutionary origins of the transmembrane porter proteins/domains are not known. Using five distinct computer programs, we here provide convincing statistical data suggesting that the transmembrane domains of ABC exporters are polyphyletic, having arisen at least three times independently. ABC1 porters arose by intragenic triplication of a primordial two-transmembrane segment (TMS)-encoding genetic element, yielding six TMS proteins. ABC2 porters arose by intragenic duplication of a dissimilar primordial three-TMS-encoding genetic element, yielding a distinctive protein family, nonhomologous to the ABC1 proteins. ABC3 porters arose by duplication of a primordial four-TMS-encoding genetic element, yielding either eight- or 10-TMS proteins. We assign each of 48 of the 50 currently recognized families of ABC exporters to one of the three evolutionarily distinct ABC types. Currently available high-resolution structural data for ABC porters are fully consistent with our findings. These results provide guides for future structural and mechanistic studies of these important transport systems.

Structural models of the human copper P-type ATPases ATP7A and ATP7B

DEFF Research Database (Denmark)

Gourdon, P.; Sitsel, Oleg; Karlsen, J.L.

2012-01-01

The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B......, allowing protein-specific properties to be addressed. Furthermore, the mapping of known disease-causing missense mutations indicates that among the heavy-metal binding domains, HMBD5 and HMBD6 are the most crucial for function, thus mimicking the single or dual HMBDs found in most copper-specific P-type...

Specific T-cell recognition of the merozoite proteins rhoptry-associated protein 1 and erythrocyte-binding antigen 1 of Plasmodium falciparum

DEFF Research Database (Denmark)

Jakobsen, P H; Hviid, L; Theander, T G

1993-01-01

The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living i...... by individuals living in an area with a high transmission rate of malaria. Most of the donor plasma samples tested contained immunoglobulin G (IgG) and IgM antibodies recognizing the merozoite proteins, while only a minority showed high IgG reactivity to the synthetic peptides.......The merozoite proteins merozoite surface protein 1 (MSP-1) and rhoptry-associated protein 1 (RAP-1) and synthetic peptides containing sequences of MSP-1, RAP-1, and erythrocyte-binding antigen 1, induced in vitro proliferative responses of lymphocytes collected from Ghanaian blood donors living...

[Importance of binding of 2,3-diphosphoglycerate and ATP to hemoglobin for erythrocyte glycolysis: activation by 2,3-diphosphoglycerate of hexokinase at intracellular conditions].

Science.gov (United States)

Geier, T; Glende, M; Reich, J G

1978-01-01

In a theoretical study the influence of hemoglobin and Mg-ions as binding partners of red cell 2,3-diphosphoglycerate and ATP was investigated. Free hemoglobin may be an efficient competitor of Mg2+ for the ligand ATP. At conditions which favour hemoglobin as binding partner (i.e. desoxygenation, low medium pH and incubation temperature, as in blood preservation) up to 95% of the whole cellular ATP (ca. 2mM in cell water) may be bound to hemoglobin (ca. 7 mM). This binding is largely prevented in the presence of physiological amounts of diphosphoglycerate (ca. 7 mM) which is in excess and has a higher binding affinity to hemoglobin. Therefore, diphosphoglycerate keeps ATP (MgATP) in cell water solution at conditions in which Hb would trop it in the presence of Mg2+ (ca. 3mM). It can be calculated that, by lack of free MgATP, the activity of hexokinase within the cell drops by a factor of greater than 10 when diphosphoglycerate is metabolized. This indirect activation by diphosphoglycerate of hexokinase is operative at free concentrations of DPG far below those which exert the well known excess inhibitory effect on hexokinase and phosphofructokinase. In a model study, the activation by diphosphoglycerate of the initial two-kinase stage was introduced into a simplified kinetic model of glycolysis. A pronounced hysteresis loop of the stationary concentrations of ATP and diphosphoglycerate was produced indicating the existence of several stationary states, one with high ATP and high diphosphoglycerate, the other one with low values. It is demonstrated that diphosphoglycerate, being a protector of glycolysis at physiological concentrations, triggers an autocatalytic breakdown of the energy state when permitted to drop to low values.

Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

International Nuclear Information System (INIS)

Fry, D.C.; Byler, D.M.; Susi, H.; Brown, M.; Kuby, S.A.; Mildvan, A.S.

1988-01-01

The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme, appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase, with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of β-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% α-helix, 38% β-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possible due to disorder, it can be fit by using methods developed on well-characterized globular proteins. The CD spectrum is best fit by assuming the presence of at most 13% α-helix in the peptide, 24 +/- 2% β-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformation changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assess by CD

Vaginal type-II mucosa is an inductive site for primary CD8+ T-cell mucosal immunity

Science.gov (United States)

Wang, Yichuan; Sui, Yongjun; Kato, Shingo; Hogg, Alison E.; Steel, Jason C.; Morris, John C.; Berzofsky, Jay A.

2014-01-01

The structured lymphoid tissues are considered the only inductive sites where primary T cell immune responses occur. The naïve T cells in structured lymphoid tissues, once being primed by antigen -bearing dendritic cells, differentiate into memory T cells and traffic back to the mucosal sites through the bloodstream. Contrary to this belief, here we show that the vaginal type-II mucosa itself, despite lack of structured lymphoid tissues, can act as an inductive site during primary CD8+ T cell immune responses. We provide evidence that the vaginal mucosa supports both the local immune priming of naïve CD8+ T cells and the local expansion of antigen-specific CD8+ T cells, thereby demonstrating a different paradigm for primary mucosal T cell immune induction. PMID:25600442

Structural models of zebrafish (Danio rerio NOD1 and NOD2 NACHT domains suggest differential ATP binding orientations: insights from computational modeling, docking and molecular dynamics simulations.

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Jitendra Maharana

Full Text Available Nucleotide-binding oligomerization domain-containing protein 1 (NOD1 and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds and Aspartic acid (Walker B formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2 interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray.

Directory of Open Access Journals (Sweden)

Daniel S Chen

2005-10-01

Full Text Available In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect.In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-gamma (IFNgamma and tumor necrosis factor-alpha (TNFalpha in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNgamma and TNFalpha did so.Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome.

Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

Science.gov (United States)

Kocher, Jacob F; Lindesmith, Lisa C; Debbink, Kari; Beall, Anne; Mallory, Michael L; Yount, Boyd L; Graham, Rachel L; Huynh, Jeremy; Gates, J Edward; Donaldson, Eric F; Baric, Ralph S

2018-05-22

Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. IMPORTANCE Caliciviruses are

Saturable binding of 35S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

International Nuclear Information System (INIS)

Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

1984-01-01

35 -S-t-Butylbicyclophosphorothionate ( 35 S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25 0 C, in 250 mM NaCl, specific binding of 35 S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. 35 S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase 3 H-GABA binding. Pharmacological studies show that 35 S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and trazolate, and a diaryl-triazine, LY81067, are also potent inhibitors of 35 S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin and LY81067 on the saturable binding of 35 S-TBPS in cortical membranes are compared. The present bindings are consistent with the interpretation that 35 S-TBPS binds, at or near the picrotoxin-sensitive anion recognition sites of the GABA/benzodiazepine/picrotoxin receptor complex

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

Science.gov (United States)

Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

Science.gov (United States)

Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance linked ATP binding cassette drug transporter ABCG2.

Science.gov (United States)

Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V

2007-12-01

Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2, which are essential for blood clotting. The naturally occurring structural analogue of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We here report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). Vitamin K3 and plumbagin inhibited the binding of [(125)I]iodoarylazidoprazosin, a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC(50) values of 7.3 and 22.6 micromol/L, respectively, but had no effect on the binding of the photoaffinity analogue to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of the ABCG2 transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared with the control cells, suggesting that they are substrates of this transporter. Collectively, these data show for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function.

ATP stimulates calcium influx in primary astrocyte cultures

International Nuclear Information System (INIS)

Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

1988-01-01

The effect of ATP and other purines on 45 Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular 45 Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 45 Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of 45 Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced 45 Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNA

International Nuclear Information System (INIS)

Hountondji, C.; Schmitter, J.M.; Beauvallet, C.; Blanquet, S.

1987-01-01

Periodate-oxidized tRNA/sup Phe/ (tRNA/sub ox//sup Phe/) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the α 2 β 2 enzyme with tRNA/sub ox//sup Phe/ results in the loss of tRNA/sup Phe/ aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[ 14 C]tRNA/sub ox//sup Phe/ covalent complex indicates that the large (α, M/sub r/ 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA/sub ox//sup Phe/. The [ 14 C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the α subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases

The O-antigen structure of bacterium Comamonas aquatica CJG.

Science.gov (United States)

Wang, Xiqian; Kondakova, Anna N; Zhu, Yutong; Knirel, Yuriy A; Han, Aidong

2017-11-01

Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1 H and 13 C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

On the mechanism of sulfite activation of chloroplast thylakoid ATPase and the relation of ADP tightly bound at a catalytic site to the binding change mechanism

Energy Technology Data Exchange (ETDEWEB)

Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

1990-01-16

Washed chloroplast thylakoid membranes upon exposure to ({sup 3}H)ADP retain in tightly bound ({sup 3}H)ADP on a catalytic site of the ATP synthase. The presence of sufficient endogenous or added Mg{sup 2+} results in an enzyme with essentially no ATPase activity. Sulfite activates the ATPase, and many molecules of ATP per synthase can be hydrolyzed before most of the bound ({sup 3}H)ADP is released, a result interpreted as indicating that the ADP is not bound at a site participating in catalysis by the sulfite-activated enzyme. The authors present evidence that this is not the case. The Mg{sup 2+}- and ADP-inhibited enzyme when exposed to MgATP and 20-100 mM sulfite shows a lag of about 1 min at 22{degree}C and of about 15 s at 37{degree}C before reaching the same steady-state rate as attained with light-activated ATPase that has not been inhibited by Mg{sup 2+} and ADP. The lag is not eliminated if the enzyme is exposed to sulfite prior to MgATP addition, indicating that ATPase turnover is necessary for the activation. The release of most of the bound ({sup 3}H)ADP parallels the onset of ATPase activity, although some ({sup 3}H)ADP is not released even with prolonged catalytic turnover and may be on poorly active or inactive enzyme or at noncatalytic sites. The results are consistent with most of the tightly bound ({sup 3}H)ADP being at a catalytic site and being replaced as this Mg{sup 2+}- and ADP-inhibited site regains equivalent participation with other catalytic sites on the activated enzyme. The sulfite activation can be explained by sulfite combination at a P{sub i} binding site of the enzyme-ADP-Mg{sup 2+} complex to give a form more readily activated by ATP binding at an alternative site.

Structural studies of a bacterial tRNA(HIS guanylyltransferase (Thg1-like protein, with nucleotide in the activation and nucleotidyl transfer sites.

Directory of Open Access Journals (Sweden)

Samantha J Hyde

Full Text Available All nucleotide polymerases and transferases catalyze nucleotide addition in a 5' to 3' direction. In contrast, tRNA(His guanylyltransferase (Thg1 enzymes catalyze the unusual reverse addition (3' to 5' of nucleotides to polynucleotide substrates. In eukaryotes, Thg1 enzymes use the 3'-5' addition activity to add G-1 to the 5'-end of tRNA(His, a modification required for efficient aminoacylation of the tRNA by the histidyl-tRNA synthetase. Thg1-like proteins (TLPs are found in Archaea, Bacteria, and mitochondria and are biochemically distinct from their eukaryotic Thg1 counterparts TLPs catalyze 5'-end repair of truncated tRNAs and act on a broad range of tRNA substrates instead of exhibiting strict specificity for tRNA(His. Taken together, these data suggest that TLPs function in distinct biological pathways from the tRNA(His maturation pathway, perhaps in tRNA quality control. Here we present the first crystal structure of a TLP, from the gram-positive soil bacterium Bacillus thuringiensis (BtTLP. The enzyme is a tetramer like human THG1, with which it shares substantial structural similarity. Catalysis of the 3'-5' reaction with 5'-monophosphorylated tRNA necessitates first an activation step, generating a 5'-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5'-end. Consistent with earlier characterization of human THG1, we observed distinct binding sites for the nucleotides involved in these two steps of activation and nucleotidyl transfer. A BtTLP complex with GTP reveals new interactions with the GTP nucleotide in the activation site that were not evident from the previously solved structure. Moreover, the BtTLP-ATP structure allows direct observation of ATP in the activation site for the first time. The BtTLP structural data, combined with kinetic analysis of selected variants, provide new insight into the role of key residues in the activation step.

Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing

DEFF Research Database (Denmark)

Werdelin, O; Mouritsen, S; Petersen, B L

1988-01-01

The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic...... fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC...... molecules are the questions discussed in this review. To us, the entire concept of processing has appeal not only because it explains some hitherto well-established, but poorly understood, phenomena such as the fact that T lymphocytes focus their attention entirely upon antigens on other cells. It has...

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

Science.gov (United States)

Martens, I; Nilsson, S A; Linder, S; Magnusson, G

1989-05-01

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.

Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

International Nuclear Information System (INIS)

Smith, L.I.

1989-01-01

The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with [ 3 H]DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both [ 3 H]DM and L-[ 35 S]methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptor function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified

The association of heavy and light chain variable domains in antibodies: implications for antigen specificity.

KAUST Repository

Chailyan, Anna

2011-06-28

The antigen-binding site of immunoglobulins is formed by six regions, three from the light and three from the heavy chain variable domains, which, on association of the two chains, form the conventional antigen-binding site of the antibody. The mode of interaction between the heavy and light chain variable domains affects the relative position of the antigen-binding loops and therefore has an effect on the overall conformation of the binding site. In this article, we analyze the structure of the interface between the heavy and light chain variable domains and show that there are essentially two different modes for their interaction that can be identified by the presence of key amino acids in specific positions of the antibody sequences. We also show that the different packing modes are related to the type of recognized antigen.

Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells

DEFF Research Database (Denmark)

Pedersen, Natasja Wulff; Chandran, P. Anoop; Qian, Yu

2017-01-01

Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide...... automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore...... laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0...

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

International Nuclear Information System (INIS)

Han, Hui; Peng, Ji-Run; Chen, Peng-Cheng; Gong, Lei; Qiao, Shi-Shi; Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua; Leng, Xi-Sheng

2011-01-01

Highlights: → Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. → An ideal artificial APCs system was successfully prepared in vivo. → Controlled release of IL-2 leads to much more T-cell expansion. → This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

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Han, Hui [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Peng, Ji-Run, E-mail: pengjr@medmail.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Chen, Peng-Cheng; Gong, Lei [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Qiao, Shi-Shi [Department of Hepatobiliary Surgery, The First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052 (China); Wang, Wen-Zhen; Cui, Zhu-Qingqing; Yu, Xin; Wei, Yu-Hua [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China); Leng, Xi-Sheng, E-mail: lengxs2003@yahoo.com.cn [Department of Hepatobiliary Surgery, Peking University People' s Hospital, Beijing 100044 (China)

2011-08-05

Highlights: {yields} Adoptive immunotherapy depends on relevant numbers of cytolytic T lymphocytes. {yields} An ideal artificial APCs system was successfully prepared in vivo. {yields} Controlled release of IL-2 leads to much more T-cell expansion. {yields} This system is better than general cellular APCs on T-cell expansion. -- Abstract: Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

Aspirin and salicylate bind to immunoglobulin heavy chain binding protein (BiP) and inhibit its ATPase activity in human fibroblasts.

Science.gov (United States)

Deng, W G; Ruan, K H; Du, M; Saunders, M A; Wu, K K

2001-11-01

Salicylic acid (SA), an endogenous signaling molecule of plants, possesses anti-inflammatory and anti-neoplastic actions in human. Its derivative, aspirin, is the most commonly used anti-inflammatory and analgesic drug. Aspirin and sodium salicylate (salicylates) have been reported to have multiple pharmacological actions. However, it is unclear whether they bind to a cellular protein. Here, we report for the first time the purification from human fibroblasts of a approximately 78 kDa salicylate binding protein with sequence identity to immunoglobulin heavy chain binding protein (BiP). The Kd values of SA binding to crude extract and to recombinant BiP were 45.2 and 54.6 microM, respectively. BiP is a chaperone protein containing a polypeptide binding site recognizing specific heptapeptide sequence and an ATP binding site. A heptapeptide with the specific sequence displaced SA binding in a concentration-dependent manner whereas a control heptapeptide did not. Salicylates inhibited ATPase activity stimulated by this specific heptapeptide but did not block ATP binding or induce BiP expression. These results indicate that salicylates bind specifically to the polypeptide binding site of BiP in human cells that may interfere with folding and transport of proteins important in inflammation.

Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma.

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Hamada, Junichi; Shoda, Katsutoshi; Masuda, Kiyoshi; Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

2016-03-29

T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC.

Merkel Cell Polyomavirus Small T Antigen Initiates Merkel Cell Carcinoma-like Tumor Development in Mice.

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Verhaegen, Monique E; Mangelberger, Doris; Harms, Paul W; Eberl, Markus; Wilbert, Dawn M; Meireles, Julia; Bichakjian, Christopher K; Saunders, Thomas L; Wong, Sunny Y; Dlugosz, Andrzej A

2017-06-15

Merkel cell carcinoma (MCC) tumor cells express several markers detected in normal Merkel cells, a nonproliferative population of neuroendocrine cells that arise from epidermis. MCCs frequently contain Merkel cell polyomavirus (MCPyV) DNA and express viral transforming antigens, sT and tLT, but the role of these putative oncogenes in MCC development, and this tumor's cell of origin, are unknown. Using a panel of preterm transgenic mice, we show that epidermis-targeted coexpression of sT and the cell fate-determinant atonal bHLH transcription factor 1 (ATOH1) leads to development of widespread cellular aggregates, with histology and marker expression mimicking that of human intraepidermal MCC. The MCC-like tumor phenotype was dependent on the FBXW7-binding domain of sT, but not the sT-PP2A binding domain. Coexpression of MCPyV tLT did not appreciably alter the phenotype driven by either sT or sT combined with ATOH1. MCPyV sT, when coexpressed with ATOH1, is thus sufficient to initiate development of epidermis-derived MCC-like tumors in mice. Cancer Res; 77(12); 3151-7. ©2017 AACR . ©2017 American Association for Cancer Research.

Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes

International Nuclear Information System (INIS)

Poli, M.A.

1985-01-01

The lipid-soluble polyether neurotoxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique receptor site associated with the voltage-sensitive sodium channel in rat brain synaptosomes. Reduction of the C 42 aldehyde function of T34 to the alcohol function of T17 using NaB 3 H 4 yielded 3 H-T17 with a specific activity of 15 Ci;/mmol. Using this specific probe, binding to sodium channels was measured at 4 0 CC, 22 0 C, and 37 0 C. Rosenthal analysis of the binding data yielded a K/sub d/ of 2.9 nM and B/sub max/ of 6.8 pmoles 3 H-T17 per mg of synaptosomal protein at 4 0 C. Both K/sub d/ and B/sub max/ were found to be temperature dependent. Depolarization of the synaptosomes by osmotic lysis resulted in the loss of 34% of the available receptor sites, with no decrease in binding affinity. Unlabeled T17, T34, and synthetic T17 (reduced T34) were equipotent in their ability to displace 3 H-T17 from its specific receptor site. Competition experiments using natural toxin probes specific for sites I-IV on the voltage-sensitive sodium channel demonstrate that 3 H-T17 does not bind to any of the previously-described neurotoxin receptor sites. A fifth site is proposed

Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes

Energy Technology Data Exchange (ETDEWEB)

Poli, M.A.

1985-01-01

The lipid-soluble polyether neurotoxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique receptor site associated with the voltage-sensitive sodium channel in rat brain synaptosomes. Reduction of the C/sub 42/ aldehyde function of T34 to the alcohol function of T17 using NaB/sup 3/H/sub 4/ yielded /sup 3/H-T17 with a specific activity of 15 Ci;/mmol. Using this specific probe, binding to sodium channels was measured at 4/sup 0/CC, 22/sup 0/C, and 37/sup 0/C. Rosenthal analysis of the binding data yielded a K/sub d/ of 2.9 nM and B/sub max/ of 6.8 pmoles /sup 3/H-T17 per mg of synaptosomal protein at 4/sup 0/C. Both K/sub d/ and B/sub max/ were found to be temperature dependent. Depolarization of the synaptosomes by osmotic lysis resulted in the loss of 34% of the available receptor sites, with no decrease in binding affinity. Unlabeled T17, T34, and synthetic T17 (reduced T34) were equipotent in their ability to displace /sup 3/H-T17 from its specific receptor site. Competition experiments using natural toxin probes specific for sites I-IV on the voltage-sensitive sodium channel demonstrate that /sup 3/H-T17 does not bind to any of the previously-described neurotoxin receptor sites. A fifth site is proposed.

Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis

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Mustafa, A S; Amoudy, H A; Wiker, H G

1998-01-01

GroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M. tuberculosis, M. tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well...... as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG). In addition, M. tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN...

Transcription factor binding sites prediction based on modified nucleosomes.

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Mohammad Talebzadeh

Full Text Available In computational methods, position weight matrices (PWMs are commonly applied for transcription factor binding site (TFBS prediction. Although these matrices are more accurate than simple consensus sequences to predict actual binding sites, they usually produce a large number of false positive (FP predictions and so are impoverished sources of information. Several studies have employed additional sources of information such as sequence conservation or the vicinity to transcription start sites to distinguish true binding regions from random ones. Recently, the spatial distribution of modified nucleosomes has been shown to be associated with different promoter architectures. These aligned patterns can facilitate DNA accessibility for transcription factors. We hypothesize that using data from these aligned and periodic patterns can improve the performance of binding region prediction. In this study, we propose two effective features, "modified nucleosomes neighboring" and "modified nucleosomes occupancy", to decrease FP in binding site discovery. Based on these features, we designed a logistic regression classifier which estimates the probability of a region as a TFBS. Our model learned each feature based on Sp1 binding sites on Chromosome 1 and was tested on the other chromosomes in human CD4+T cells. In this work, we investigated 21 histone modifications and found that only 8 out of 21 marks are strongly correlated with transcription factor binding regions. To prove that these features are not specific to Sp1, we combined the logistic regression classifier with the PWM, and created a new model to search TFBSs on the genome. We tested the model using transcription factors MAZ, PU.1 and ELF1 and compared the results to those using only the PWM. The results show that our model can predict Transcription factor binding regions more successfully. The relative simplicity of the model and capability of integrating other features make it a superior method

The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.

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Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

2011-06-17

The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.

MHC class II-derived peptides can bind to class II molecules, including self molecules, and prevent antigen presentation

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Rosloniec, E F; Vitez, L J; Buus, S

1990-01-01

the alpha k-3 peptide binds slightly less well. These combined data, suggesting that class II-derived peptides can bind to MHC class II molecules, including the autologous molecule from which they are derived, have important implications for the molecular basis of alloreactivity and autoreactivity. Further...... found in the first and third polymorphic regions (PMR) of the A alpha k chain (alpha k-1 and alpha k-3) were capable of inhibiting the presentation of three different HEL-derived peptide antigens to their appropriate T cells. In addition, the alpha k-1 peptide inhibited the presentation of the OVA(323......-339) immunodominant peptide to the I-Ad-restricted T cell hybridomas specific for it. Prepulsing experiments demonstrated that the PMR peptides were interacting with the APC and not with the T cell hybridomas. These observations were confirmed and extended by the demonstration that the alpha k-1 and alpha k-3...

Opposing effects of estradiol- and testosterone-membrane binding sites on T47D breast cancer cell apoptosis

International Nuclear Information System (INIS)

Kampa, Marilena; Nifli, Artemissia-Phoebe; Charalampopoulos, Ioannis; Alexaki, Vassilia-Ismini; Theodoropoulos, Panayiotis A.; Stathopoulos, Efstathios N.; Gravanis, Achille; Castanas, Elias

2005-01-01

Classical steroid mode of action involves binding to intracellular receptors, the later acting as ligand-activated nuclear transcription factors. Recently, membrane sites for different steroids have been also identified, mediating rapid, non-genomic, steroid actions. Membrane sites for estrogen and androgen have been found in a number of different cell types, bearing or not classical intracellular receptors. In the present study, with the use of radioligand binding, flow cytometry and confocal laser microscopy, we report that T47D human breast cancer cells express specific and saturable membrane receptors for both estrogen (K D 4.06 ± 3.31 nM) and androgen (K D 7.64 ± 3.15 nM). Upon activation with BSA-conjugated, non-permeable ligands (E 2 -BSA and testosterone-BSA), membrane estrogen receptors protect cells from serum-deprivation-induced apoptosis, while androgen receptors induce apoptosis in serum-supplemented T47D cells. In addition, co-incubation of cells with a fixed concentration of one steroid and varying concentrations of the other reversed the abovementioned effect (apoptosis for androgen, and anti-apoptosis for E 2 ), suggesting that the fate of the cell depends on the relative concentration of either steroid in the culture medium. We also report the identification of membrane receptors for E 2 and androgen in biopsy slides from breast cancer patients. Both sites are expressed, with the staining for membrane E 2 being strongly present in ER-negative, less differentiated, more aggressive tumors. These findings suggest that aromatase inhibitors may exert their beneficial effects on breast cancer by also propagating the metabolism of local steroids towards androgen, inducing thus cell apoptosis through membrane androgen receptor activation

Isolation of a peptide binding protein and its role in antigen presentation

International Nuclear Information System (INIS)

Lakey, E.; Pierce, S.K.; Margoliash, E.

1986-01-01

A mouse T cell hybrid, TPc9.1, recognizes pigeon cytochrome c (Pc) as processed and presented by histocompatible antigen presenting cells (APC). When paraformaldehyde fixed APC are employed, only a peptide fragment of Pc, Pc 81-104, and not the native Pc, is capable of stimulating TPc9.1 cells. Pc 81-104 appears to associate tightly with the APC surface since paraformaldehyde fixed APC which have been incubated with Pc 81-104 remain stimulatory following extensive washing. When APC are surface labeled with 125 I, solubilized and affinity purified on Pc 81-104-Sepharose 4B columns, two predominant polypeptides of approximately 72 and 74 kd are isolated. Little or no immunoglobulin, Class I or Class II proteins are obtained under these conditions. Antisera from rabbits immunized with the affinity purified material, but not preimmune sera, block the activation of TPc 9.1 cells by Pc as well as Pc 81-104 when presented by live APC. Furthermore, these antisera are even more effective in blocking the activation of TPc9.1 cells by either APC which had been pulsed with Pc and then paraformaldehyde fixed, or by Pc 81-104 when added to paraformaldehyde fixed APC, suggesting that these antisera were not affecting antigen processing. Thus, these peptide binding proteins may play a role in antigen presentation, and they are being further characterized

Characteristics of the formation of enzyme-bound ATP from medium inorganic phosphate by mitochondrial F1 adenosinetriphosphatase in the presence of dimethyl sulfoxide

International Nuclear Information System (INIS)

Kandpal, R.P.; Stempel, K.E.; Boyer, P.D.

1987-01-01

Addition of dimethyl sulfoxide promotes the formation of enzyme-bound ATP from medium P/sub i/ by mitochondrial F 1 adenosinetriphosphatase that has tightly bound ADP present. Measurements are reported of medium P/sub i/ ↔ H 18 OH exchange and of the dependence of formation of enzyme-bound ATP on P/sub i/ concentration. Attainment of an apparent equilibrium between medium P/sub i/ and bound ATP requires longer than 30 min, even though the rates of P/sub i/ binding and release after apparent equilibrium is reached would suffice for a faster approach to equilibrium. Slow protein conformational changes or other unknown modulating factors may be responsible for the slow rate of bound ATP formation. After apparent equilibrium is reached, each P/sub i/ that binds to the enzyme reversibly forms ATP about 50 times before being released to the medium. The rate of interconversion of bound ATP to bound ADP and P/sub i/ is much slower than that in the absence of dimethyl sulfoxide as measured with sufficient low ATP concentrations so that single-site catalysis is favored. Although the interconversion rate is slowed, the equilibrium constant for bound ATP formation from bound ADP and P/sub i/ is not far from unity. Dimethyl sulfoxide favors the formation of enzyme-bound ATP by promoting the competent binding of P/sub i/ to enzyme with ADP bound at a catalytic site rather than by promoting formation of bound ATP from bound ADP and P/sub i/

Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti-liver-kidney microsome type-1 antibodies (LKM1).

Science.gov (United States)

Yamamoto, A M; Cresteil, D; Boniface, O; Clerc, F F; Alvarez, F

1993-05-01

Anti-liver-kidney microsome type-1 antibodies (LKM1), present in sera from a group of patients with autoimmune hepatitis, are directed against P450IID6. Previous work, using cDNA constructions spanning most of the P450IID6 protein defined the main immunogenic site between the amino acids (aa), 254-271 and predicted the presence of other putative immunogenic sites in the molecule. Fusion proteins from new cDNA constructions, spanning so-far-untested regions between aa 1-125 and 431-522, were not recognized by LKM1-positive sera. Synthetic peptides, representing sequences from putative immunogenic regions or previously untested regions, allowed a precise definition of four antigenic sites located between peptides 257-269, 321-351, 373-389 and 410-429, which were recognized, respectively, by 14, 8, 1 and 2 out of 15 LKM1-positive sera tested. The minimal sequence of the main antigenic site (peptide 257-269) recognized by the autoantibody was established to be WDPAQPPRD (peptide 262-270). In addition, deletion and replacement experiments showed that aa 263 (Asp) was essential for the binding of the autoantibody to peptide 262-270. Analysis of the second most frequently recognized peptide between aa 321-351, was performed using peptides 321-339 and 340-351 in competitive inhibition studies. Complete elimination of antibody binding to peptide 321-351 obtained by absorption of both shorter peptides indicated that peptide 321-351 is a discontinuous antigenic site. LKM1-positive sera reacting against peptide 321-351 recognized either both the shorter peptides or just one of them preferentially. Results of the present study suggest that the production of LKM1 antibodies is an antigen-driven, poly- or oligoclonal B cell response. The identification of antigenic sites will allow: (i) the development of specific diagnostic tests and (ii) further studies on the pathogenic value of LKM1 antibodies in autoimmune hepatitis.

Recognition of microbial glycolipids by Natural Killer T cells

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Dirk Michael Zajonc

2015-08-01

Full Text Available T cells can recognize microbial antigens when presented by dedicated antigen-presenting molecules. While peptides are presented by classical members of the Major Histocompatibility (MHC family (MHC I and II, lipids, glycolipids and lipopeptides can be presented by the non-classical MHC member CD1. The best studied subset of lipid-reactive T cells are Type I Natural killer T (iNKT cells that recognize a variety of different antigens when presented by the non-classical MHCI homolog CD1d. iNKT cells have been shown to be important for the protection against various microbial pathogens, including B. burgdorferi the causative agents of Lyme disease and S. pneumoniae, which causes pneumococcal meningitis and community-acquired pneumonia. Both pathogens carry microbial glycolipids that can trigger the T cell antigen receptor (TCR, leading to iNKT cell activation. iNKT cells have an evolutionary conserved TCR alpha chain, yet retain the ability to recognize structurally diverse glycolipids. They do so using a conserved recognition mode, in which the TCR enforces a conserved binding orientation on CD1d. TCR binding is accompanied by structural changes within the TCR binding site of CD1d, as well as the glycolipid antigen itself. In addition to direct recognition of microbial antigens, iNKT cells can also be activated by a combination of cytokines (IL-12/IL-18 and TCR stimulation. Many microbes carry TLR antigens and microbial infections can lead to TLR activation. The subsequent cytokine response in turn lower the threshold of TCR mediated iNKT cell activation, especially when weak microbial or even self-antigens are presented during the cause of the infection. In summary, iNKT cells can be directly activated through TCR triggering of strong antigens, while cytokines produced by the innate immune response may be necessary for TCR triggering and iNKT cell activation in the presence of weak antigens. Here we will review the molecular basis of iNKT cell

Mouse ATP-Binding Cassette (ABC) Transporters Conferring Multi-Drug Resistance

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Shuaizhang, L I; Zhang, Wen; Yin, Xuejiao; Xing, Shilai; Xie, Qunhui; Cao, Zhengyu; Zhao, Bin

2015-04-28

The ABC (ATP-binding cassette) transporter is one of the largest and most ancient protein families with members functioning from protozoa to human. The resistance of cancer and tumor cells to anticancer drugs is due to the over-expression of some ABC transporters, which may finally lead to chemotherapy failure. The mouse ABC transporters are classified into seven subfamilies by phylogenetic analysis. The mouse ABC transporter gene, alias, chromosomal location and function have been determined. Within the ABC super-family, the MDR transporters (Abcb1, Abcc1, Abcg2) in mouse models have been proved to be valuable to investigate the biochemistry and physiological functions. This review concentrates on the multidrug resistance of mouse ABC transporters in cancer and tumor cells.

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

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Philip Serwer

2017-05-01

Full Text Available Adenosine triphosphate (ATP cleavage powers packaging of a double-stranded DNA (dsDNA molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.

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Grupp, Stephan A; Kalos, Michael; Barrett, David; Aplenc, Richard; Porter, David L; Rheingold, Susan R; Teachey, David T; Chew, Anne; Hauck, Bernd; Wright, J Fraser; Milone, Michael C; Levine, Bruce L; June, Carl H

2013-04-18

Chimeric antigen receptor-modified T cells with specificity for CD19 have shown promise in the treatment of chronic lymphocytic leukemia (CLL). It remains to be established whether chimeric antigen receptor T cells have clinical activity in acute lymphoblastic leukemia (ALL). Two children with relapsed and refractory pre-B-cell ALL received infusions of T cells transduced with anti-CD19 antibody and a T-cell signaling molecule (CTL019 chimeric antigen receptor T cells), at a dose of 1.4×10(6) to 1.2×10(7) CTL019 cells per kilogram of body weight. In both patients, CTL019 T cells expanded to a level that was more than 1000 times as high as the initial engraftment level, and the cells were identified in bone marrow. In addition, the chimeric antigen receptor T cells were observed in the cerebrospinal fluid (CSF), where they persisted at high levels for at least 6 months. Eight grade 3 or 4 adverse events were noted. The cytokine-release syndrome and B-cell aplasia developed in both patients. In one child, the cytokine-release syndrome was severe; cytokine blockade with etanercept and tocilizumab was effective in reversing the syndrome and did not prevent expansion of chimeric antigen receptor T cells or reduce antileukemic efficacy. Complete remission was observed in both patients and is ongoing in one patient at 11 months after treatment. The other patient had a relapse, with blast cells that no longer expressed CD19, approximately 2 months after treatment. Chimeric antigen receptor-modified T cells are capable of killing even aggressive, treatment-refractory acute leukemia cells in vivo. The emergence of tumor cells that no longer express the target indicates a need to target other molecules in addition to CD19 in some patients with ALL.

Carbohydrates as T-cell antigens with implications in health and disease.

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Sun, Lina; Middleton, Dustin R; Wantuch, Paeton L; Ozdilek, Ahmet; Avci, Fikri Y

2016-10-01

Glycosylation is arguably the most ubiquitous post-translational modification on proteins in microbial and mammalian cells. During the past few years, there has been intensive research demonstrating that carbohydrates, either in pure forms or in conjunction with proteins or lipids, evoke and modulate adaptive immune responses. We now know that carbohydrates can be directly recognized by T cells or participate in T-cell stimulation as components of T-cell epitopes. T-cell recognition of carbohydrate antigens takes place via their presentation by major histocompatibility complex pathways on antigen-presenting cells. In this review, we summarize studies on carbohydrates as T-cell antigens modulating adaptive immune responses. Through discussion of glycan-containing antigens, such as glycoproteins, glycolipids, zwitterionic polysaccharides and carbohydrate-based glycoconjugate vaccines, we will illustrate the key molecular and cellular interactions between carbohydrate antigens and T cells and the implications of these interactions in health and disease. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

LIBRA: LIgand Binding site Recognition Application.

Science.gov (United States)

Hung, Le Viet; Caprari, Silvia; Bizai, Massimiliano; Toti, Daniele; Polticelli, Fabio

2015-12-15

In recent years, structural genomics and ab initio molecular modeling activities are leading to the availability of a large number of structural models of proteins whose biochemical function is not known. The aim of this study was the development of a novel software tool that, given a protein's structural model, predicts the presence and identity of active sites and/or ligand binding sites. The algorithm implemented by ligand binding site recognition application (LIBRA) is based on a graph theory approach to find the largest subset of similar residues between an input protein and a collection of known functional sites. The algorithm makes use of two predefined databases for active sites and ligand binding sites, respectively, derived from the Catalytic Site Atlas and the Protein Data Bank. Tests indicate that LIBRA is able to identify the correct binding/active site in 90% of the cases analyzed, 90% of which feature the identified site as ranking first. As far as ligand binding site recognition is concerned, LIBRA outperforms other structure-based ligand binding sites detection tools with which it has been compared. The application, developed in Java SE 7 with a Swing GUI embedding a JMol applet, can be run on any OS equipped with a suitable Java Virtual Machine (JVM), and is available at the following URL: http://www.computationalbiology.it/software/LIBRAv1.zip. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

Science.gov (United States)

Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

2002-12-24

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

Chimeric antigen receptor T-cell therapy for solid tumors

Directory of Open Access Journals (Sweden)

Kheng Newick

2016-01-01

Full Text Available Chimeric antigen receptor (CAR T cells are engineered constructs composed of synthetic receptors that direct T cells to surface antigens for subsequent elimination. Many CAR constructs are also manufactured with elements that augment T-cell persistence and activity. To date, CAR T cells have demonstrated tremendous success in eradicating hematological malignancies (e.g., CD19 CARs in leukemias. This success is not yet extrapolated to solid tumors, and the reasons for this are being actively investigated. Here in this mini-review, we discuss some of the key hurdles encountered by CAR T cells in the solid tumor microenvironment.

Radioimmunoassay for Epstein-Barr Virus (EBV)-associated Nuclear Antigen (EBNA). Binding of iodinated antibodies to antigen immobilized in polyacrylamide gel

International Nuclear Information System (INIS)

Dolken, G.; Klein, G.

1977-01-01

A solid-phase radioimmunoassay was developed for the EBV-associated nuclear antigen (EBNA). Total homogenates of EBV-DNA and EBNA positive or negative cells were polymerized in polyacrylamide gel and compared for their ability to bind 125 I-IgG prepared from anti-EBNA positive and anti-EBNA negative sera. EBNA specific binding was demonstrated and confirmed by serological and cellular specificity controls. The assay allows the quantitation of antigen or antibody even in the presence of detergents and is suitable for biochemical characterization of the antigen. Reciprocal blocking studies with extracts from different cell lines showed quantitative and qualitative differences. One part of the EBNA specificiti(es) present in the human Burkitt lymphoma derived lines RAJI, DAUDI and AW-RAMOS was lacking in B96-8, a marmoset line carrying EBV derived from a human infectious mononucleosis line. This result may reflect differences in the viral genomes derived from Burkitt lymphoma and infectious mononucleosis lines or differences in the host cells. (author)

Antigen delivery by α2-macroglobulin enhances the cytotoxic T lymphocyte response

Science.gov (United States)

Bowers, Edith V.; Horvath, Jeffrey J.; Bond, Jennifer E.; Cianciolo, George J.; Pizzo, Salvatore V.

2009-01-01

α2M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, α2M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that α2M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that α2M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA257–264 (SIINFEKL) demonstrated up to 25-fold greater IFN-γ and IL-2 secretion when treated in vitro with α2M*-OVA compared with soluble OVA. The frequency of IFN-γ-producing cells was increased ∼15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by α2M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α2M*-OVA. These α2M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that α2M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support α2M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing. PMID:19652028

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.

Science.gov (United States)

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.

Adaptive evolution of transcription factor binding sites

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Berg Johannes

2004-10-01

Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

Science.gov (United States)

Ll, Juan Modole; Cabrer, Bartolomé; Parmeggiani, Andrea; Azquez, David V

1971-01-01

Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G. PMID:4331558

Conventional CD4+ T cells present bacterial antigens to induce cytotoxic and memory CD8+ T cell responses.

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Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Osuna-Pérez, Jesús; Torres-Torresano, Mónica; Zorita, Virgina; Martínez-Riaño, Ana; Boccasavia, Viola; Borroto, Aldo; Martínez Del Hoyo, Gloria; González-Granado, José María; Alarcón, Balbino; Sánchez-Madrid, Francisco; Veiga, Esteban

2017-11-17

Bacterial phagocytosis and antigen cross-presentation to activate CD8 + T cells are principal functions of professional antigen presenting cells. However, conventional CD4 + T cells also capture and kill bacteria from infected dendritic cells in a process termed transphagocytosis (also known as transinfection). Here, we show that transphagocytic T cells present bacterial antigens to naive CD8 + T cells, which proliferate and become cytotoxic in response. CD4 + T-cell-mediated antigen presentation also occurs in vivo in the course of infection, and induces the generation of central memory CD8 + T cells with low PD-1 expression. Moreover, transphagocytic CD4 + T cells induce protective anti-tumour immune responses by priming CD8 + T cells, highlighting the potential of CD4 + T cells as a tool for cancer immunotherapy.

Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

Science.gov (United States)

Denys, A; Allain, F; Foxwell, B; Spik, G

1997-08-01

Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

Science.gov (United States)

Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

2016-02-01

P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp. Published by Elsevier Inc.

Bitopic Ligands and Metastable Binding Sites

DEFF Research Database (Denmark)

Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

2017-01-01

of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

Genome-wide identification of whole ATP-binding cassette (ABC) transporters in the intertidal copepod Tigriopus japonicus.

Science.gov (United States)

Jeong, Chang-Bum; Kim, Bo-Mi; Lee, Jae-Seong; Rhee, Jae-Sung

2014-08-05

The ATP-binding cassette (ABC) transporter superfamily is one of the largest transporter gene families and is observed in all animal taxa. Although a large set of transcriptomic data was recently assembled for several species of crustaceans, identification and annotation of the large ABC transporter gene family have been very challenging. In the intertidal copepod Tigriopus japonicus, 46 putative ABC transporters were identified using in silico analysis, and their full-length cDNA sequences were characterized. Phylogenetic analysis revealed that the 46 T. japonicus ABC transporters are classified into eight subfamilies (A-H) that include all the members of all ABC subfamilies, consisting of five ABCA, five ABCB, 17 ABCC, three ABCD, one ABCE, three ABCF, seven ABCG, and five ABCH subfamilies. Of them, unique isotypic expansion of two clades of ABCC1 proteins was observed. Real-time RT-PCR-based heatmap analysis revealed that most T. japonicus ABC genes showed temporal transcriptional expression during copepod development. The overall transcriptional profile demonstrated that half of all T. japonicus ABC genes were strongly associated with at least one developmental stage. Of them, transcripts TJ-ABCH_88708 and TJ-ABCE1 were highly expressed during all developmental stages. The whole set of T. japonicus ABC genes and their phylogenetic relationships will provide a better understanding of the comparative evolution of essential gene family resources in arthropods, including the crustacean copepods.

GPU-Based Point Cloud Superpositioning for Structural Comparisons of Protein Binding Sites.

Science.gov (United States)

Leinweber, Matthias; Fober, Thomas; Freisleben, Bernd

2018-01-01

In this paper, we present a novel approach to solve the labeled point cloud superpositioning problem for performing structural comparisons of protein binding sites. The solution is based on a parallel evolution strategy that operates on large populations and runs on GPU hardware. The proposed evolution strategy reduces the likelihood of getting stuck in a local optimum of the multimodal real-valued optimization problem represented by labeled point cloud superpositioning. The performance of the GPU-based parallel evolution strategy is compared to a previously proposed CPU-based sequential approach for labeled point cloud superpositioning, indicating that the GPU-based parallel evolution strategy leads to qualitatively better results and significantly shorter runtimes, with speed improvements of up to a factor of 1,500 for large populations. Binary classification tests based on the ATP, NADH, and FAD protein subsets of CavBase, a database containing putative binding sites, show average classification rate improvements from about 92 percent (CPU) to 96 percent (GPU). Further experiments indicate that the proposed GPU-based labeled point cloud superpositioning approach can be superior to traditional protein comparison approaches based on sequence alignments.

GTP plus water mimics ATP in the active site of protein kianse CK2

DEFF Research Database (Denmark)

Niefind, K; Pütter, M; Guerra, B

1999-01-01

The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently...... use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs...

[Stabilization of Cadmium Contaminated Soils by Ferric Ion Modified Attapulgite (Fe/ATP)--Characterizations and Stabilization Mechanism].

Science.gov (United States)

Rong, Yang; Li, Rong-bo; Zhou, Yong-li; Chen, Jing; Wang, Lin-ling; Lu, Xiao-hua

2015-08-01

Ferric ion modified attapulgite (Fe/ATP) was prepared by impregnation and its structure and morphology were characterized. The toxicity characteristic leaching procedure (TCLP) was used to evaluate the effect of Cadmium( Cd) stabilization in soil with the addition of attapulgite (ATP) and Fe/ATP. The stabilization mechanism of Cd was further elucidated by comparing the morphologies and structure of ATP and Fe/ATP before and after Cd adsorption. Fe/ATP exhibited much better adsorption capacity than ATP, suggesting different adsorption mechanisms occurred between ATP and Fe/ATP. The leaching concentrations of Cd in soil decreased by 45% and 91% respectively, with the addition of wt. 20% ATP and Fe/ATP. The former was attributed to the interaction between Cd2 and --OH groups by chemical binding to form inner-sphere complexes in ATP and the attachment between Cd2+ and the defect sites in ATP framework. Whereas Cd stabilization with Fe/ATP was resulted from the fact that the active centers (--OH bonds or O- sites) on ATP could react with Fe3+ giving Fe--O--Cd-- bridges, which helped stabilize Cd in surface soil. What'more, the ferric oxides and metal hydroxides on the surface of ATP could interact with Cd, probably by the formation of cadmium ferrite. In conclusion, Fe/ATP, which can be easily prepared, holds promise as a potential low-cost and environmental friendly stabilizing agent for remediation of soil contaminated with heavy metals.

Skin-Resident T Cells Drive Dermal Dendritic Cell Migration in Response to Tissue Self-Antigen.

Science.gov (United States)

Ali, Niwa; Zirak, Bahar; Truong, Hong-An; Maurano, Megan M; Gratz, Iris K; Abbas, Abul K; Rosenblum, Michael D

2018-05-01

Migratory dendritic cell (DC) subsets deliver tissue Ags to draining lymph nodes (DLNs) to either initiate or inhibit T cell-mediated immune responses. The signals mediating DC migration in response to tissue self-antigen are largely unknown. Using a mouse model of inducible skin-specific self-antigen expression, we demonstrate that CD103 + dermal DCs (DDCs) rapidly migrate from skin to skin DLN (SDLNs) within the first 48 h after Ag expression. This window of time was characterized by the preferential activation of tissue-resident Ag-specific effector T cells (Teffs), with no concurrent activation of Ag-specific Teffs in SDLNs. Using genetic deletion and adoptive transfer approaches, we show that activation of skin-resident Teffs is required to drive CD103 + DDC migration in response to tissue self-antigen and this Batf3-dependent DC population is necessary to mount a fulminant autoimmune response in skin. Conversely, activation of Ag-specific Teffs in SDLNs played no role in DDC migration. Our studies reveal a crucial role for skin-resident T cell-derived signals, originating at the site of self-antigen expression, to drive DDC migration during the elicitation phase of an autoimmune response. Copyright © 2018 by The American Association of Immunologists, Inc.

ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

Science.gov (United States)

ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg2+

International Nuclear Information System (INIS)

Du, Z.; Boyer, P.D.

1989-01-01

The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg 2+ with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg 2+ is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg 2+ released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg 2+ the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg 2+ and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg 2+ . The replacement of [ 3 H]ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg 2+ and ADP bound at one catalytic site without P i block catalysis by all three enzyme sites. The inhibited form induced by Mg 2+ and ADP may occur only under laboratory conditions and not have an in vivo role

Staphylococcal enterotoxin-A directly stimulates signal transduction and interferon-gamma production in psoriatic T-cell lines

DEFF Research Database (Denmark)

Nielsen, M B; Odum, N; Gerwien, J

1998-01-01

class II. Here we address the question of whether SEA can directly activate psoriatic T cells in the absence of professional antigen-presenting cells. We show that SEA induces i) tyrosine phosphorylation of several proteins, ii) downregulation of the T-cell receptor (TCR), and iii) production......-mediated proliferation. In contrast, SEA with a mutation in the MHC class II alpha-binding site induces IFN-gamma and a qualitatively changed tyrosine phosphorylation profile. Both mutations delete the co-stimulatory effect on cytokine-mediated proliferation. This suggests that both MHC class II binding sites...

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

Directory of Open Access Journals (Sweden)

Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Solution structure of the 45-residue ATP-binding peptide of adenylate kinase as determined by 2-D NMR, FTIR, and CD spectroscopy

International Nuclear Information System (INIS)

Fry, D.C.; Byler, D.M.; Susi, H.; Brown, E.M.; Kuby, S.A.; Mildyan, A.S.

1986-01-01

In the X-ray structure of adenylate kinase residues 1-45 exist as 47% α-helix, 29% β-structure (strands and turns) and 24% coil. The solution structure of a synthetic peptide corresponding to residues 1-45, which constitutes the MgATP binding site was studied by 3 independent spectroscopic methods. Globularity of the peptide was shown by its broad NMR resonances which narrow upon denaturation, and by its ability to bind MgATP with similar affinity and conformation as the intact enzyme does. COSY and NOESY NMR methods at 250 and 500 MHz reveal proximities among NH, Cα, and Cβ protons indicative of >20% α-helix, and >20% β-structure. Correlation of regions of secondary structure with the primary sequence by 2D NMR indicates at least one α-helix (res. 23 to 29) and two β-strands (res. 12 to 15 and 34 to 38). The broad amide I band in the deconvoluted FTIR spectrum could be fit as the sum of 4 peaks due to specific secondary structures, yielding ≤=45% α-helix, ≤=40% β-structure and ≥=15% coil. The CD spectrum, from 185-250 nm, interpreted with a 3-parameter basis set, yielded 20 +/- 5% α=helix, and ≤=20% β-structure. The solution structure of peptide 1-45 thus approximates that of residues 1-45 in the crystal

A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

DEFF Research Database (Denmark)

Kongsbak, Kristine Grønning; Bergmann, Rikke; Sørensen, Pernille Louise

2013-01-01

We present a full-length a1b2c2 GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate......-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode...... of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines a1T206 and c2T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important a1H101 and the N-methyl group near a1Y159, a1T206, and a1Y209. We present a binding mode...

The binding sites for cocaine and dopamine in the dopamine transporter overlap

DEFF Research Database (Denmark)

Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L

2008-01-01

Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog Leu......T. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed...... inhibition of dopamine transport by cocaine....

Double-lock ratchet mechanism revealing the role of  SER-344 in FoF1 ATP synthase

KAUST Repository

Beke-Somfai, T.

2011-03-07

In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP+Pi→ATP+H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the γP-OADP bond-forming step via a double-proton transfer. At this step, the highly conserved αS344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.

Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme

International Nuclear Information System (INIS)

Kasho, V.N.; Boyer, P.D.

1989-01-01

Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F 1 , F 0 -ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F 1 ,F 0 -ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and P i as medium ATP concentration was lowered was determined by 18 O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the P i formed as ATP concentration is decreased to the micromolar range. The F 1 ,F 0 -ATPase from neurospora mitochondria showed an event more pronounced modulation, similar to that of other F 1 -type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F 1 ,F 0 -ATPases

Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

Science.gov (United States)

Farb, Joshua N; Morrical, Scott W

2009-01-16

Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

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Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Splice Site Mutations in the ATP7A Gene

DEFF Research Database (Denmark)

Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

2011-01-01

Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...... to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations...

Antigen presentation by resting B cells. Radiosensitivity of the antigen-presentation function and two distinct pathways of T cell activation

International Nuclear Information System (INIS)

Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

1984-01-01

In this report we have examined the ability of small resting B cells to act as antigen-presenting cells (APC) to antigen-specific MHC-restricted T cells as assessed by either T cell proliferation or T cell-dependent B cell stimulation. We found that 10 of 14 in vitro antigen-specific MHC-restricted T cell clones and lines and three of four T cell hybridomas could be induced to either proliferate or secrete IL-2 in the presence of lightly irradiated (1,000 rads) purified B cells and the appropriate foreign antigen. All T cell lines and hybridomas were stimulated to proliferate or make IL-2 by macrophage- and dendritic cell-enriched populations and all T cells tested except one hybridoma caused B cell activation when stimulated with B cells as APC. Furthermore, lightly irradiated, highly purified syngeneic B cells were as potent a source of APC for inducing B cell activation as were low density dendritic and macrophage-enriched cells. Lymph node T cells freshly taken from antigen-primed animals were also found to proliferate when cultured with purified B cells and the appropriate antigen. This APC function was easily measured when the cells were irradiated with 1,000 rads, but was greatly diminished or absent when they were irradiated with 3,300 rads. In addition, this radiosensitivity allowed us to easily distinguish B cell antigen presentation from presentation by the dendritic cell and macrophage, as the latter was resistant to 3,300 rads. Finally, one T cell clone that failed to proliferate when B cells were used as APC was able to recruit allogeneic B cells to proliferate in the presence of syngeneic B cells and the appropriate antigen. This result suggests that there are at least two distinct pathways of activation in T cells, one that leads to T cell proliferation and one that leads to the secretion of B cell recruitment factor(s)

Viral sequestration of antigen subverts cross presentation to CD8(+ T cells.

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Eric F Tewalt

2009-05-01

Full Text Available Virus-specific CD8(+ T cells (T(CD8+ are initially triggered by peptide-MHC Class I complexes on the surface of professional antigen presenting cells (pAPC. Peptide-MHC complexes are produced by two spatially distinct pathways during virus infection. Endogenous antigens synthesized within virus-infected pAPC are presented via the direct-presentation pathway. Many viruses have developed strategies to subvert direct presentation. When direct presentation is blocked, the cross-presentation pathway, in which antigen is transferred from virus-infected cells to uninfected pAPC, is thought to compensate and allow the generation of effector T(CD8+. Direct presentation of vaccinia virus (VACV antigens driven by late promoters does not occur, as an abortive infection of pAPC prevents production of these late antigens. This lack of direct presentation results in a greatly diminished or ablated T(CD8+ response to late antigens. We demonstrate that late poxvirus antigens do not enter the cross-presentation pathway, even when identical antigens driven by early promoters access this pathway efficiently. The mechanism mediating this novel means of viral modulation of antigen presentation involves the sequestration of late antigens within virus factories. Early antigens and cellular antigens are cross-presented from virus-infected cells, as are late antigens that are targeted to compartments outside of the virus factories. This virus-mediated blockade specifically targets the cross-presentation pathway, since late antigen that is not cross-presented efficiently enters the MHC Class II presentation pathway. These data are the first to describe an evasion mechanism employed by pathogens to prevent entry into the cross-presentation pathway. In the absence of direct presentation, this evasion mechanism leads to a complete ablation of the T(CD8+ response and a potential replicative advantage for the virus. Such mechanisms of viral modulation of antigen presentation

Two classes of ouabain binding sites in ferret heart and two forms of Na+-K+-ATPase

Energy Technology Data Exchange (ETDEWEB)

Ng, Y.C.; Akera, T.

1987-05-01

In partially purified Na+-K+-adenosinetriphosphatase (ATPase) obtained from ferret heart, ouabain produced a monophasic inhibition curve; however, the curve spanned over 5 logarithmic units, indicating the presence of more than one classes of enzyme. (/sup 3/H)ouabain binding studies revealed high-and low-affinity binding sites in approximately equal abundance, with apparent dissociation constants of 10 and 230 nM, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphoenzyme formed from (gamma-/sup 32/P)ATP showed two distinct K+-sensitive bands of approximately 100,000 molecular weight. Phosphoenzyme formation from the high-molecular-weight alpha(+) form was selectively inhibited by N-ethylmaleimide. Ouabain caused a 50% inhibition of phosphorylation of the alpha(+) form at 40 nM and the lower-molecular-weight alpha form at 300 nM. In papillary muscle preparations, 1-30 nM ouabain produced a modest positive inotropic effect that reached an apparent plateau at 30 nM. Further increases in ouabain concentrations, however, produced additional and prominent inotropic effects at 0.1-10 microM. These results indicate for the first time in cardiac muscle that the high- and low-affinity ouabain binding sites are associated with the alpha(+) and alpha forms of the Na+-K+-ATPase, respectively, and that binding of ouabain to either of these sites causes enzyme inhibition and the positive inotropic effect.

Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

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Yan Li

2015-04-01

Full Text Available Cyclin-dependent kinase 2 (CDK2 is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP binding site (Site I and two non-competitive binding sites (Site II and III. In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV. All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate. In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2.

Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide

NARCIS (Netherlands)

Villen, J.; Borras, E.; Schaaper, W.M.M.; Meloen, R.H.; Davila, M.; Domingo, E.; Giralt, E.; Andreu, D.

2002-01-01

Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

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Brand, M D; Lehninger, A L

1977-01-01

The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems. PMID:17116

A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

Science.gov (United States)

Foda, Zachariah H; Shan, Yibing; Kim, Eric T; Shaw, David E; Seeliger, Markus A

2015-01-20

Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

Science.gov (United States)

Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

1997-03-15

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes

Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation

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Kuo-Ching Sheng

2013-01-01

Full Text Available Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

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Gomez Bianca P

2012-10-01

Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

Science.gov (United States)

Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

2017-01-27

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of praziquantel on the differential expression of mouse hepatic genes and parasite ATP binding cassette transporter gene family members during Schistosoma mansoni infection.

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Melissa C Sanchez

2017-06-01

Full Text Available Schistosomiasis is a chronic parasitic disease caused by sexually dimorphic blood flukes of the genus Schistosoma. Praziquantel (PZQ is the only drug widely available to treat the disease but does not kill juvenile parasites. Here we report the use of next generation sequencing to study the transcriptional effect of PZQ on murine hepatic inflammatory, immune and fibrotic responses to Schistosoma mansoni worms and eggs. An initial T helper cell 1 (Th1 response is induced against schistosomes in mice treated with drug vehicle (Vh around the time egg laying begins, followed by a T helper cell 2 (Th2 response and the induction of genes whose action leads to granuloma formation and fibrosis. When PZQ is administered at this time, there is a significant reduction in egg burden yet the hepatic Th1, Th2 and fibrotic responses are still observed in the absence of granuloma formation suggesting some degree of gene regulation may be induced by antigens released from the dying adult worms. Quantitative real-time PCR was used to examine the relative expression of 16 juvenile and adult S. mansoni genes during infection and their response to Vh and PZQ treatment in vivo. While the response of stress genes in adult parasites suggests the worms were alive immediately following exposure to PZQ, they were unable to induce transcription of any of the 9 genes encoding ATP-binding cassette (ABC transporters tested. In contrast, juvenile schistosomes were able to significantly induce the activities of ABCB, C and G family members, underscoring the possibility that these efflux systems play a major role in drug resistance.

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

Science.gov (United States)

Naguib, Fardos N. M.; Rais, Reem H.; Al Safarjalani, Omar N.; el Kouni, Mahmoud H.

2015-01-01

Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ± 0.0002 mM, KATP = 0.05 ± 0.008 mM, and Vmax = 920 ± 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5′-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii. PMID:26112826

Chimeric antigen receptor T cell (CAR-T) immunotherapy for solid tumors: lessons learned and strategies for moving forward.

Science.gov (United States)

Li, Jian; Li, Wenwen; Huang, Kejia; Zhang, Yang; Kupfer, Gary; Zhao, Qi

2018-02-13

Recently, the US Food and Drug Administration (FDA) approved the first chimeric antigen receptor T cell (CAR-T) therapy for the treatment CD19-positive B cell acute lymphoblastic leukemia. While CAR-T has achieved remarkable success in the treatment of hematopoietic malignancies, whether it can benefit solid tumor patients to the same extent is still uncertain. Even though hundreds of clinical trials are undergoing exploring a variety of tumor-associated antigens (TAA), no such antigen with comparable properties like CD19 has yet been identified regarding solid tumors CAR-T immunotherapy. Inefficient T cell trafficking, immunosuppressive tumor microenvironment, suboptimal antigen recognition specificity, and lack of safety control are currently considered as the main obstacles in solid tumor CAR-T therapy. Here, we reviewed the solid tumor CAR-T clinical trials, emphasizing the studies with published results. We further discussed the challenges that CAR-T is facing for solid tumor treatment and proposed potential strategies to improve the efficacy of CAR-T as promising immunotherapy.

Detection of Avian Antigen-Specific T Cells Induced by Viral Vaccines

DEFF Research Database (Denmark)

Dalgaard, Tina Sørensen; Norup, Liselotte Rothmann; Juul-Madsen, Helle Risdahl

2016-01-01

Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen. There is ......Live attenuated viral vaccines are widely used in commercial poultry production, but the development of new effective inactivated/subunit vaccines is needed. Studies of avian antigen-specific T cells are primarily based on analyses ex vivo after activating the cells with recall antigen...

Blockade of Extracellular ATP Effect by Oxidized ATP Effectively Mitigated Induced Mouse Experimental Autoimmune Uveitis (EAU.

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Ronglan Zhao

Full Text Available Various pathological conditions are accompanied by ATP release from the intracellular to the extracellular compartment. Extracellular ATP (eATP functions as a signaling molecule by activating purinergic P2 purine receptors. The key P2 receptor involved in inflammation was identified as P2X7R. Recent studies have shown that P2X7R signaling is required to trigger the Th1/Th17 immune response, and oxidized ATP (oxATP effectively blocks P2X7R activation. In this study we investigated the effect of oxATP on mouse experimental autoimmune uveitis (EAU. Our results demonstrated that induced EAU in B6 mice was almost completely abolished by the administration of small doses of oxATP, and the Th17 response, but not the Th1 response, was significantly weakened in the treated mice. Mechanistic studies showed that the therapeutic effects involve the functional change of a number of immune cells, including dendritic cells (DCs, T cells, and regulatory T cells. OxATP not only directly inhibits the T cell response; it also suppresses T cell activation by altering the function of DCs and Foxp3+ T cell. Our results demonstrated that inhibition of P2X7R activation effectively exempts excessive autoimmune inflammation, which may indicate a possible therapeutic use in the treatment of autoimmune diseases.

Association of ATP-Binding Cassette Transporter A1 Gene Polymorphisms in Type 2 Diabetes Mellitus among Malaysians

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Polin Haghvirdizadeh

2015-01-01

Full Text Available Background. Type 2 diabetes mellitus (T2DM is a complex polygenic disorder characterized by impaired insulin resistance, insulin secretion, and dysregulation of lipid and protein metabolism with environmental and genetic factors. ATP-binding cassette transporter A1 (ABCA1 gene polymorphisms are reported as the one of the genetic risk factors for T2DM in various populations with conflicting results. This study was conducted based on PCR-HRM to determine the frequency of ABCA1 gene by rs2230806 (R219K, rs1800977 (C69T, and rs9282541 (R230C polymorphisms Malaysian subjects. Methods. A total of 164 T2DM and 165 controls were recruited and their genotypes for ABCA1 gene polymorphisms were determined based on the real time high resolution melting analysis. Results. There was a significant difference between the subjects in terms of age, BMI, FPG, HbA1c, HDL, LDL, and TG P<0.05. There was a significant association between HOM of R219K P=0.005, among Malaysian subjects; moreover, allele frequency revealed the significant difference in A allele of R219K P=0.003. But, there was no significant difference in genotypic and allelic frequencies of C69T and R230C polymorphism. Conclusion. R219K polymorphism of ABCA1 gene can be considered as a genetic risk factor for T2DM subjects among Malaysians.

Control of ATP hydrolysis by ADP bound at the catalytic site of chloroplast ATP synthase as related to protonmotive force and Mg sup 2+

Energy Technology Data Exchange (ETDEWEB)

Du, Z.; Boyer, P.D. (Univ. of California, Los Angeles (USA))

1989-01-24

The activation of the ATP synthesis and hydrolysis capacity of isolated chloroplast membranes by protonmotive force is known to be associated with the release of tightly bound ADP from the ATP synthase. The data support the view that the activation requires only those structural changes occurring in the steady-state reaction mechanism. The trapping of ADP released during light activation or the chelation of Mg{sup 2+} with EDTA effectively reduces the rate of decay of the ATPase activity. When the release of tightly bound ADP and Mg{sup 2+} is promoted by light activation, followed by immediate dilution and washing to retard the rebinding of the ADP and Mg{sup 2+} released, the ATPase activity remains high in the dark long after the protonmotive force has disappeared. After the addition of ADP and Mg{sup 2+} the decay of the ATPase activity has the same characteristics as those of the unwashed chloroplast membrane. The results are interpreted as indicating that both Mg{sup 2+} and ADP must be present prior to exposure to MgATP for the ATPase to be inhibited. However, in contrast to the isolated chloroplast ATPase, the steady-state activity of the membrane-bound ATPase is not inhibited by excess Mg{sup 2+}. The replacement of ({sup 3}H)ADP from catalytic sites during hydrolysis of unlabeled ATP or during photophosphorylation with unlabeled ADP occurs as anticipated if Mg{sup 2+} and ADP bound at one catalytic site without P{sub i} block catalysis by all three enzyme sites. The inhibited form induced by Mg{sup 2+} and ADP may occur only under laboratory conditions and not have an in vivo role.

Chimeric Antigen Receptor-Engineered T Cells in Tumor Immunotherapy: From Bench to Beside

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Peng WANG

2017-06-01

Full Text Available Chimeric antigen receptor-engineered T cells (CAR-T cells, a classification of cultured T cells after modification of gene engineering technology, can recognize specific tumor antigens in a major histocompatibility complex (MHC-independent manner, consequently leading to the activation of antitumor function. The recent studies have confirmed that a variety of tumor-associated antigens (TAAs can act as target antigens for CAR-T cells. Nowadays, CAR T-cell therapy, one of the most potential tumor immunotherapies, has made great breakthroughs in hematological malignancies and promising outcomes in solid tumors. In this article, the biological characteristics and antitumor mechanism of CAR-T cells, and their application in tumor treatment were mainly reviewed.

The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Javid-Majd, Farah; Yang, Dong [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States); Ioerger, Thomas R. [Department of Computer Science, Texas A& M University, College Station, Texas 77843-2128 (United States); Sacchettini, James C., E-mail: sacchett@tamu.edu [Department of Biochemistry and Biophysics, Texas A& M University, College Station, Texas 77843-2128 (United States)

2008-06-01

The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.

Two distinct affinity binding sites for IL-1 on human cell lines

International Nuclear Information System (INIS)

Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

1989-01-01

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

Towards Deciphering the Hidden Mechanisms That Contribute to the Antigenic Activation Process of Human Vγ9Vδ2 T Cells

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Lola Boutin

2018-04-01

Full Text Available Vγ9Vδ2 T cells represent a major unconventional γδ T cell subset located in the peripheral blood of adults in humans and several non-human primates. Lymphocytes that constitute this transitional subset can sense subtle level changes of intracellular phosphorylated intermediates of the isoprenoid biosynthesis pathway (phosphoantigens, pAg, such as isopentenyl pyrophosphate, during cell stress events. This unique antigenic activation process operates in a rigorous framework that requires the expression of butyrophilin 3A1 (BTN3A1/CD277 molecules, which are type I glycoproteins that belong to the B7 family. Several studies have further shown that pAg specifically bind to the intracellular B30.2 domain of BTN3A1 linked to the antigenic activation of Vγ9Vδ2 T cells. Here, we highlight the recent advances in BTN3A1 dynamics induced upon the binding of pAg and the contribution of the different subunits to this activation process. Recent reports support that conformational modifications of BTN3A1 might represent a key step in the detection of infection or tumorigenesis by Vγ9Vδ2 T cells. A better understanding of this mechanism will help optimize novel immunotherapeutical approaches that target defined functions of this unique γδ T cell subset.

Leukemia-associated antigens in man.

Science.gov (United States)

Brown, G; Capellaro, D; Greaves, M

1975-12-01

Rabbit antisera raised against acute lymphoblastic leukemia (ALL) cells were used to distinguish ALL from other leukemias, to identify rare leukemia cells in the bone marrow of patients in remission, and to define human leukemia-associated antigens. Antibody binding was studied with the use of immunofluorescence reagents and the analytic capacity of the Fluorescence Activated Cell Sorter-1 (FACS-1). The results indicated that most non-T-cell ALL have three leukemia-associated antigens on their surface which are absent from normal lymphoid cells: 1) an antigen shared with myelocytes, myeloblastic leukemia cells, and fetal liver (hematopoietic) cells; 2) an antigen shared with a subset of intermediate normoblasts in normal bone marrow and fetal liver; and 3) an antigen found thus far only on non-T-cell ALL and in some acute undifferentiated leukemias, which we therefore regard as a strong candidate for a leukemia-specific antigen. These antigens are absent from a subgroup of ALL patients in which the lymphoblasta express T-cell surface markers. Preliminary studies on the bone marrow samples of patients in remission indicated that rare leukemia cells were present in some samples. The implications of these findings with respect to the heterogeneity and cell origin(s) of ALL, its diagnosis, and its potential monitoring during treatment were discussed.

Functional antigen binding by the defective B cells of CBA/N mice.

Science.gov (United States)

Snippe, H; Merchant, B; Lizzio, E F; Inman, J K

1982-01-01

CBA/N mice have an X-linked B cell defect which prevents them from responding to nonmitogenic thymic independent (TI-2) antigens such as dinitrophenylated DNP-Ficoll (1,2). The F1 male progeny of CBA/N female mice express the same defect. Spleen cell suspensions from such defective mice (CBA/N X C3H/HeN F1 males) could not respond to DNP-Ficoll following in vitro immunization and subsequent transfer into irradiated, syngeneic, F1 male recipients as expected. In contrast, normal CBA/N X C3H/HeN F1 female spleen cells could respond and effect a "rescue"; they mounted strong plaque-forming cell responses 7 days after in vitro exposure to DNP-Ficoll and subsequent transfer into irradiated F1 male recipients. Defective F1 male spleen cells, however, could bind significant quantities of 125I-DNP-Ficoll after in vitro exposure. Extensive washing of these spleen cells could not reverse this binding. Such DNP-Ficoll-exposed and washed F1 male spleen cells could, after transfer, aid normal untreated F1 female cells in their rescue function. The defective F1 male spleen cells could convey immunogenic quantities of DNP-Ficoll to the "rescuing" F1 female cells. Mitomycin treatment of F1 male cells did not interfere with their conveyor function. Goat anti-mouse mu serum impeded the passive antigen conveyor function of defective F1 male cells as did prior exposure to high concentrations of free DNP hapten. Our data support the view that the B cell defect of CBA/N X C3H/HeN F1 male mice does not relate to antigen binding, but rather to an inability to be effectively triggered by certain cell-bound polymeric antigens.

Performance and specificity of the covalently linked immunomagnetic separation-ATP method for rapid detection and enumeration of enterococci in coastal environments.

Science.gov (United States)

Zimmer-Faust, Amity G; Thulsiraj, Vanessa; Ferguson, Donna; Jay, Jennifer A

2014-05-01

The performance and specificity of the covalently linked immunomagnetic separation-ATP (Cov-IMS/ATP) method for the detection and enumeration of enterococci was evaluated in recreational waters. Cov-IMS/ATP performance was compared with standard methods: defined substrate technology (Enterolert; IDEXX Laboratories), membrane filtration (EPA Method 1600), and an Enterococcus-specific quantitative PCR (qPCR) assay (EPA Method A). We extend previous studies by (i) analyzing the stability of the relationship between the Cov-IMS/ATP method and culture-based methods at different field sites, (ii) evaluating specificity of the assay for seven ATCC Enterococcus species, (iii) identifying cross-reacting organisms binding the antibody-bead complexes with 16S rRNA gene sequencing and evaluating specificity of the assay to five nonenterococcus species, and (iv) conducting preliminary tests of preabsorption as a means of improving the assay. Cov-IMS/ATP was found to perform consistently and with strong agreement rates (based on exceedance/compliance with regulatory limits) of between 83% and 100% compared to the culture-based Enterolert method at a variety of sites with complex inputs. The Cov-IMS/ATP method is specific to five of seven different Enterococcus spp. tested. However, there is potential for nontarget bacteria to bind the antibody, which may be reduced by purification of the IgG serum with preabsorption at problematic sites. The findings of this study help to validate the Cov-IMS/ATP method, suggesting a predictable relationship between the Cov-IMS/ATP method and traditional culture-based methods, which will allow for more widespread application of this rapid and field-portable method for coastal water quality assessment.

Evidence of two different Na/sup +/-dependent (/sup 3/H)-ouabain binding sites of a Na/sup +/-K/sup +/-ATPase of guinea-pig hearts

Energy Technology Data Exchange (ETDEWEB)

Fricke, U; Klaus, W [Koeln Univ. (Germany, F.R.)

1977-11-01

The influence of various Na/sup +/ concentrations on (/sup 3/H)-ouabain binding was studied in experiments on a microsomal Na/sup +/-K/sup +/-adenosine triphosphatase (ATPase) from guinea-pig hearts. The ATP-independent cardiac glycoside binding was not influenced by increasing Na/sup +/ concentrations. However, a good correlation was found between the ATP-dependent (/sup 3/H)-ouabain binding and Na/sup +/ concentration. A more detailed analysis of these results revealed two distinct processes involved in this interaction: one ouabain binding process was activated at rather low Na/sup +/ concentrations, (Ksub(0.5) = 4.5 mM); this type of (/sup 3/H)-ouabain binding was strongly correlated to the Na/sup +/ concentration necessary for half maximum phosphorylation (Ksub(0.5) = 1 mM). The other ouabain binding process was predominant at high Na/sup +/ concentrations (Ksub(0.5 = 69 mM). On the basis of the commonly accepted ATPase reaction cycle a model for the interaction of cardiac glycosides with the Na/sup +/-K/sup +/-ATPase is proposed, assuming two different binding sites for cardiac glycosides (E/sub 2/ -P and Esub(l) -P) and involving a translocation of these drugs from an outer to an inner compartment of the cell membrane.

The antibody response of pregnant Cameroonian women to VAR2CSA ID1-ID2a, a small recombinant protein containing the CSA-binding site

DEFF Research Database (Denmark)

Babakhanyan, Anna; Leke, Rose G F; Salanti, Ali

2014-01-01

In pregnant women, Plasmodium falciparum-infected erythrocytes expressing the VAR2CSA antigen bind to chondroitin sulfate A in the placenta causing placental malaria. The binding site of VAR2CSA is present in the ID1-ID2a region. This study sought to determine if pregnant Cameroonian women natura...

Characterization of a human antigen specific helper factor

International Nuclear Information System (INIS)

Richardson, B.

1986-01-01

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with 35 S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEM line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants

A negative feedback modulator of antigen processing evolved from a frameshift in the cowpox virus genome.

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Jiacheng Lin

2014-12-01

Full Text Available Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.

Chimeric antigen receptor T cells: a novel therapy for solid tumors