Journal: Frontiers in Immunology

Article Title: A Peptide-Based PD1 Antagonist Enhances T-Cell Priming and Efficacy of a Prophylactic Malaria Vaccine and Promotes Survival in a Lethal Malaria Model

doi: 10.3389/fimmu.2020.01377

Figure Lengend Snippet: LD01 increases antigen-specific CD8 + T cell numbers following AdPyCS vaccination. (A) At day 12 post-AdPyCS immunization, immunogenicity was assessed by measuring the number of splenic PyCS-specific, IFN-γ-secreting CD8 + T cells using the ELISpot assay after stimulation with the H-2kd restricted CD8 epitope SYVPSAEQI. Data are expressed as the mean ± SD. Data from one of two independent experiments are shown. Significant differences between AdPyCS + control peptide and treated mice were determined using a one-way ANOVA multiple comparisons test and are denoted by * p < 0.05 and ** p < 0.005. (B) Immunized and treated mice were challenged with Py 17XNL sporozoites IV. Data represent two independent experiments ( n = 10/group/experiment) and are expressed as the mean percent protection. Parasitemia was assessed beginning at day 3 post-challenge, and sterile protection was defined as the absence of parasite detection in the blood through day 7 by microscopic examination of Giemsa-stained thin smears.

Article Snippet: The relative numbers of splenic PyCS-specific, IFN-γ-secreting CD8 + T cells of AdPyCS- or RAS-immunized mice were determined by an ELISpot assay, using a mouse IFN-γ ELISpot kit (Abcam, Cambridge, MA) and a synthetic 9-mer peptide, SYVPSAEQI (Peptide 2.0 Inc., Chantilly, VA) corresponding to the immunodominant CD8 + T cell epitope within PyCS, as previously described ( ).

Techniques: Enzyme-linked Immunospot, Staining

Journal: Frontiers in Immunology

Article Title: A Peptide-Based PD1 Antagonist Enhances T-Cell Priming and Efficacy of a Prophylactic Malaria Vaccine and Promotes Survival in a Lethal Malaria Model

doi: 10.3389/fimmu.2020.01377

Figure Lengend Snippet: LD01 enhances antigen-specific CD8 + T cell numbers following RAS vaccination. At day 12 post-RAS immunization, immunogenicity was assessed by measuring the number of splenic PyCS-specific, IFN-γ-secreting CD8 + T cells using the ELISpot assay after stimulation with the H-2kd restricted CD8 epitope SYVPSAEQI. Data are expressed as the mean ± SD. Data from one of two independent experiments are shown. Significant differences between RAS alone and treated mice were determined using a two-tailed Unpaired t -test and are denoted by ** p < 0.005.

Article Snippet: The relative numbers of splenic PyCS-specific, IFN-γ-secreting CD8 + T cells of AdPyCS- or RAS-immunized mice were determined by an ELISpot assay, using a mouse IFN-γ ELISpot kit (Abcam, Cambridge, MA) and a synthetic 9-mer peptide, SYVPSAEQI (Peptide 2.0 Inc., Chantilly, VA) corresponding to the immunodominant CD8 + T cell epitope within PyCS, as previously described ( ).

Techniques: Enzyme-linked Immunospot, Two Tailed Test

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Modular organization of human ErbB4 and YAP2 proteins. (a) ErbB4 contains the canonical ECD-TM-ICD receptor tyrosine kinase modular cassette, where the central single-helical transmembrane (TM) domain is flanked between an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain (ICD). The three PPXY motifs (designated PY1, PY2 and PY3) within the ICD are located at the extreme C-terminus. Note that the amino acid sequence of 12-mer peptides containing the PPXY motifs and flanking residues are provided. The numerals indicate the nomenclature used in this study to distinguish residues within and flanking the PPXY motifs relative to the first consensus proline, which is arbitrarily assigned zero. (b) YAP2 is comprised of a tandem copy of WW domains, designated WW1 and WW2, located N-terminal to the trans-activation (TA) domain.

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Sequencing, Activation Assay

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Representative ITC isotherms for the binding of WW1 domain of YAP2 to ErbB4_PY1 (a), ErbB4_PY2 (b) and ErbB4_PY3 (c) peptides. The upper panels show the raw ITC data expressed as change in thermal power with respect to time over the period of titration. In the lower panels, change in molar heat is expressed as a function of molar ratio of corresponding peptide to WW1 domain. The red solid lines in the lower panels show the fit of data to a one-site binding model using the integrated ORIGIN software as described earlier [22, 20].

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Titration, Software

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Thermodynamic parameters for the binding of WW1 and WW2 domains of YAP2 to PPXY peptides derived from the ICD of ErbB4

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Derivative Assay, Sequencing

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Thermodynamic parameters for the binding of WW1 domain of YAP2 to wildtype (PY3_WT) and single alanine mutants of ErbB4_PY3 peptide

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Sequencing

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Thermodynamic parameters for the binding of WW2 domain of YAP2 to wildtype (PY3_WT) and single alanine mutants of ErbB4_PY3 peptide

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay, Sequencing

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Far-UV CD spectra of ErbB4_PY1 (red), ErbB4_PY2 (green) and ErbB4_PY3 (blue) peptides. Note that the mean ellipticity, [θ], was calculated using Eq [3].

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Circular Dichroism

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Structural models of WW1 (a) and WW2 (b) domains of YAP2 in complex with ErbB4_PY3 peptide containing the PPXY motif. The β-strands in the WW domains are shown in blue with loops depicted in gray and the peptide is colored yellow. Note that two orientations related by a 90°-rotation about the horizontal axis are depicted for the inquisitive eye. The sidechain moieties of all residues, including the PPXY motif (which corresponds to P0, P+1 and Y+3), within the bound peptide are shown in green. For the WW domains, the sidechain moieties colored in red denote all residues pointing toward the peptide on the concave side.

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques:

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Conformational dynamics as probed through MD simulations conducted on WW1 and WW2 domains of YAP2 in complex with ErbB4_PY3 peptide containing the PPXY motif. (a) RMSD of backbone atoms (N, Cα and C) within each simulated structure relative to the initial modeled structure of WW1 (top panel) and WW2 (bottom panel) domains in complex with ErbB4_PY3 peptide as a function of simulation time. Note that the overall RMSD for each WW-peptide complex (black) is deconvoluted into the WW domain alone (red) and the peptide alone (green). (b) RMSF of backbone atoms (N, Cα and C) averaged over the entire course of corresponding MD trajectory of WW1 (top panel) and WW2 (bottom panel) domains in complex with ErbB4_PY3 peptide as a function of residue number within each WW domain. The shaded vertical rectangular boxes denote residues located within the β1-β2 and β2-β3 loops. (c) RMSF of backbone atoms (N, Cα and C) averaged over the entire course of corresponding MD trajectory of WW1 (top panel) and WW2 (bottom panel) domains in complex with ErbB4_PY3 peptide as a function of residue number within the peptide (see Figure 1a for nomenclature). The PPXY motif and the flanking residues are overlayed for reference.

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Residue

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Intermolecular distances, as probed through MD simulations, between consensus residues within the PPXY motif of ErbB4_PY3 peptide and residues lining the binding groove within WW1 and WW2 domains of YAP2. (a) Distance between Cγ pyrrolidine carbon of P0 within the PPXY motif and Nε1 indole nitrogens of W199 and W258 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (b) Distance between Cγ pyrrolidine carbon of P+1 within the PPXY motif and Cζ phenolic carbons of Y188 and Y247 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (c) Distance between Oη phenolic oxygen of Y+3 within the PPXY motif and Nδ1 imidazole nitrogens of H192 and H251 located respectively within WW1 (top panel) and WW2 (bottom panel) domains.

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay

Journal: Biochimie

Article Title: Molecular Basis of the Binding of YAP Transcriptional Regulator to the ErbB4 Receptor Tyrosine Kinase

doi: 10.1016/j.biochi.2014.01.011

Figure Lengend Snippet: Intermolecular distances, as probed through MD simulations, between residues flanking the PPXY motif of ErbB4_PY3 peptide and residues lining the binding groove within WW1 and WW2 domains of YAP2. (a) Distance between Cγ1/Cγ2 methyl carbons of V-3 within the PPXY motif and Cδ1/Cδ2 indole carbons of W199 and W258 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (b) Distance between Cδ1/Cδ2 methyl carbons of L-2 within the PPXY motif and Cδ carbons of Q186 and E245 located respectively within WW1 (top panel) and WW2 (bottom panel) domains. (c) Distance between Nη1/Nη2 guanidine nitrogens of R+6 within the PPXY motif and Oε1/Oε2 carbonyl oxygens of E178 and E237 located respectively within WW1 (top panel) and WW2 (bottom panel) domains.

Article Snippet: Peptide synthesis 12-mer wildtype and mutant peptides spanning various PPXY motifs within the ICD of human ErbB4 were commercially obtained from GenScript Corporation.

Techniques: Binding Assay