Receptor binding and escape from Beta antibody responses drive Omicron-B.1.1.529 evolution

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced. Omicron contains a total of 30 substitutions plus deletions and an insertion in Spike, far more than any previously reported variant. The mutations include those previously identified by In-vitro evolution to contribute to high-affinity binding to ACE2, including mutations Q498R and N501Y critical in forming additional interactions in the interface. Together with increased charge complementarity between the RBD and ACE2, these substantially increase affinity and potentially virus transmissibility through increased syncytia formation. Further mutations promote immune evasion. We have studied the binding of a large panel of potent monoclonal antibodies generated from early pandemic or Beta infected cases. Mutations in Omicron will likely compromise the binding of many of these and additionally, the binding of antibodies under commercial development, however residual binding should provide protection from severe disease.

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Shark Attack: High affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation

Journal of Biotechnology ◽

10.1016/j.jbiotec.2014.04.023 ◽

2014 ◽

Vol 191 ◽

pp. 236-245 ◽

Cited By ~ 43

Author(s):

Stefan Zielonka ◽

Niklas Weber ◽

Stefan Becker ◽

Achim Doerner ◽

Andreas Christmann ◽

...

Keyword(s):

Binding Proteins ◽

Affinity Maturation ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity

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Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells.

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3415 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3415-3424 ◽

Cited By ~ 42

Author(s):

W M Kavanaugh ◽

A Klippel ◽

J A Escobedo ◽

L T Williams

Keyword(s):

Tyrosine Phosphorylation ◽

Platelet Derived Growth Factor ◽

Affinity Binding ◽

Pdgf Receptor ◽

Phosphatidylinositol 3 Kinase ◽

High Affinity Binding ◽

High Affinity ◽

Pdgf Stimulation ◽

Stimulation Of

The activated platelet-derived growth factor (PDGF) receptor physically associates with p85, a subunit of phosphatidylinositol-3 kinase. Although this interaction may activate phosphatidylinositol-kinase and is crucial for PDGF-induced mitogenesis, it has not been shown whether p85 is modified in the process. p85 contains two SH2 (Src homology) domains, designated SH2-N and SH2-C. Recent experiments have shown that the SH2-C domain alone determines high-affinity binding of p85 to the PDGF receptor. The function of SH2-N, which binds receptors with lower affinity, is unknown. In this study, using a receptor-blotting technique, we find that p85 is modified by PDGF stimulation of intact cells. This modification involves inhibition of binding of the SH2-N region of p85 to the PDGF receptor. Studies with vanadate suggest that tyrosine phosphorylation of p85 is responsible for the modification of p85 detected by receptor blotting. Furthermore, recombinant p85 is modified in a similar manner when it is tyrosine phosphorylated in vitro by PDGF receptors. Tyrosine phosphorylation of p85 does not block binding of the SH2-C domain and therefore does not release p85 from high-affinity binding sites on the receptor in vitro. Instead, phosphorylation may regulate the ability of the SH2-N of p85 to bind to a different portion of the PDGF receptor or to another molecule in the signaling complex. This study provides the first evidence that p85 is tyrosine phosphorylated upon PDGF stimulation of cells and suggests that tyrosine phosphorylation of p85 regulates its activity or its interaction with other proteins.

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Fluorine-18 Labeled Fluorofuranylnorprogesterone ([18F]FFNP) and Dihydrotestosterone ([18F]FDHT) Prepared by “Fluorination on Sep-Pak” Method

Molecules ◽

10.3390/molecules24132389 ◽

2019 ◽

Vol 24 (13) ◽

pp. 2389 ◽

Cited By ~ 2

Author(s):

Falguni Basuli ◽

Xiang Zhang ◽

Burchelle Blackman ◽

Margaret E. White ◽

Elaine M. Jagoda ◽

...

Keyword(s):

Androgen Receptors ◽

Quantitative Measure ◽

Emission Tomography ◽

In Vitro Assays ◽

Affinity Binding ◽

Synthesis Time ◽

High Affinity ◽

Molar Activity ◽

Positron Emission

To further explore the scope of our recently developed “fluorination on Sep-Pak” method, we prepared two well-known positron emission tomography (PET) tracers 21-[18F]fluoro-16α,17α-[(R)-(1′-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione furanyl norprogesterone ([18F]FFNP) and 16β-[18F]fluoro-5α-dihydrotestosterone ([18F]FDHT). Following the “fluorination on Sep-Pak” method, over 70% elution efficiency was observed with 3 mg of triflate precursor of [18F]FFNP. The overall yield of [18F]FFNP was 64–72% (decay corrected) in 40 min synthesis time with a molar activity of 37–81 GBq/µmol (1000–2200 Ci/mmol). Slightly lower elution efficiency (~55%) was observed with the triflate precursor of [18F]FDHT. Fluorine-18 labeling, reduction, and deprotection to prepare [18F]FDHT were performed on Sep-Pak cartridges (PS-HCO3 and Sep-Pak plus C-18). The overall yield of [18F]FDHT was 25–32% (decay corrected) in 70 min. The molar activity determined by using mass spectrometry was 63–148 GBq/µmol (1700–4000 Ci/mmol). Applying this quantitative measure of molar activity to in vitro assays [18F]FDHT exhibited high-affinity binding to androgen receptors (Kd~2.5 nM) providing biological validation of this method.

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The Arg-Gly-Asp–Containing, Solvent-Exposed Loop of Ptr ToxA Is Required for Internalization

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-3-0315 ◽

2008 ◽

Vol 21 (3) ◽

pp. 315-325 ◽

Cited By ~ 55

Author(s):

Viola A. Manning ◽

Sara M. Hamilton ◽

P. Andrew Karplus ◽

Lynda M. Ciuffetti

Keyword(s):

Phosphorylation Site ◽

Affinity Binding ◽

Mesophyll Cells ◽

High Affinity ◽

Treatment Zone ◽

Ptr Toxa ◽

High Affinity Binding Site ◽

Toxin Uptake

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 × 106 per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.

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Phosphorylation and DNA binding of the regulator DcuR of the fumarate-responsive two-component system DcuSR of Escherichia coli

Microbiology ◽

10.1099/mic.0.26900-0 ◽

2004 ◽

Vol 150 (4) ◽

pp. 877-883 ◽

Cited By ~ 20

Author(s):

Ingo G. Janausch ◽

Inma Garcia-Moreno ◽

Daniela Lehnen ◽

Yvonne Zeuner ◽

Gottfried Unden

Keyword(s):

Escherichia Coli ◽

Binding Site ◽

Response Regulator ◽

Sensory System ◽

Phosphoryl Transfer ◽

Affinity Binding ◽

High Affinity ◽

Two Component ◽

Target Promoters

The function of the response regulator DcuR of the DcuSR fumarate two-component sensory system of Escherichia coli was analysed in vitro. Isolated DcuR protein was phosphorylated by the sensory histidine kinase, DcuS, and ATP, or by acetyl phosphate. In gel retardation assays with target promoters (frdA, dcuB, dctA), phosphoryl DcuR (DcuR-P) formed a high-affinity complex, with an apparent K D (app. K D) of 0·2–0·3 μM DcuR-P, and a low-affinity (app. K D 0·8–2 μM) complex. The high-affinity complex was formed only with promoters transcriptionally-regulated by DcuSR, whereas low-affinity binding was seen also with some DcuSR-independent promoters. The binding site of DcuR-P at the dcuB promoter was determined by DNase I footprinting. One binding site of 42–52 nt (position −359 to −400/−410 nt upstream of the transcriptional start) was identified in the presence of low and high concentrations of DcuR-P. Non-phosphorylated DcuR, or DcuR-D56N mutated in the phosphoryl-accepting Asp56 residue, showed low-affinity binding to target promoters. DcuR-D56N was still able to interact with DcuS. DcuR-D56N increased the phosphorylation of DcuS and competitively inhibited phosphoryl transfer to wild-type DcuR.

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CDK9 Autophosphorylation Regulates High-Affinity Binding of the Human Immunodeficiency Virus Type 1 Tat–P-TEFb Complex to TAR RNA

Molecular and Cellular Biology ◽

10.1128/mcb.20.18.6958-6969.2000 ◽

2000 ◽

Vol 20 (18) ◽

pp. 6958-6969 ◽

Cited By ~ 109

Author(s):

Mitchell E. Garber ◽

Timothy P. Mayall ◽

Eric M. Suess ◽

Jill Meisenhelder ◽

Nancy E. Thompson ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

Immunodeficiency Virus ◽

Tar Rna ◽

Cdk9 Phosphorylation ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathioneS-transferase–C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat–P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1–728)], but not truncated CycT1(1–303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1–303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1–303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat–P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

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An alphavirus replicon-derived candidate vaccine against Rift Valley fever virus

Epidemiology and Infection ◽

10.1017/s0950268808001696 ◽

2009 ◽

Vol 137 (9) ◽

pp. 1309-1318 ◽

Cited By ~ 37

Author(s):

M. T. HEISE ◽

A. WHITMORE ◽

J. THOMPSON ◽

M. PARSONS ◽

A. A. GROBBELAAR ◽

...

Keyword(s):

Rift Valley ◽

Rift Valley Fever ◽

Rift Valley Fever Virus ◽

Fluorescent Antibody ◽

Severe Disease ◽

Neutralizing Antibody ◽

Fever Virus ◽

Antibody Responses ◽

Valley Fever

SUMMARYRift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus (genusPhlebovirus) associated with severe disease in livestock and fatal encephalitis or haemorrhagic fever in a proportion of infected humans. Although live attenuated and inactivated vaccines have been used in livestock, and on a limited scale in humans, there is a need for improved anti-RVFV vaccines. Towards this goal, Sindbis virus replicon vectors expressing the RVFV Gn and Gc glycoproteins, as well as the non-structural nsM protein, were constructed and evaluated for their ability to induce protective immune responses against RVFV. These replicon vectors were shown to produce the RVFV glycoproteins to high levelsin vitroand to induce systemic anti-RVFV antibody responses in immunized mice, as determined by RVFV-specific ELISA, fluorescent antibody tests, and demonstration of a neutralizing antibody response. Replicon vaccination also provided 100% protection against lethal RVFV challenge by either the intraperitoneal or intranasal route. Furthermore, preliminary results indicate that the replicon vectors elicit RVFV-specific neutralizing antibody responses in vaccinated sheep. These results suggest that alphavirus-based replicon vectors can induce protective immunity against RVFV, and that this approach merits further investigation into its potential utility as a RVFV vaccine.

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Specific high-affinity binding of 125I-labeled mouse interferon to interferon resistant embryonal carcinoma cells in vitro

Virology ◽

10.1016/0042-6822(81)90241-5 ◽

1981 ◽

Vol 114 (2) ◽

pp. 585-588 ◽

Cited By ~ 40

Author(s):

Michel Aguet ◽

Ion Gresser ◽

Ara G. Hovanessian ◽

Marie-Thérèse Bandu ◽

Brigitte Blanchard ◽

...

Keyword(s):

Embryonal Carcinoma ◽

Carcinoma Cells ◽

Affinity Binding ◽

Embryonal Carcinoma Cells ◽

High Affinity Binding ◽

High Affinity

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Zinc Trafficking 1. Probing the Roles of Proteome, Metallothionein, and Glutathione

Metallomics ◽

10.1093/mtomcs/mfab055 ◽

2021 ◽

Author(s):

Afsana Mahim ◽

Mohammad Mahim ◽

David H Petering

Keyword(s):

Carbonic Anhydrase ◽

Binding Sites ◽

Affinity Binding ◽

Conditional Stability ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Conditional Stability Constant ◽

High Affinity Binding ◽

High Affinity

Abstract The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that non-specific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione. In agreement, inclusion of glutathione accelerated the reaction in a concentration-dependent manner. The implications of abundant high affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with glutathione in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

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