scholarly journals Identifying protein–protein interactions in somatic hypermutation

2005 ◽  
Vol 201 (4) ◽  
pp. 493-496 ◽  
Author(s):  
Myron F. Goodman ◽  
Matthew D. Scharff
Keyword(s):  
Mouse Model ◽  
Protein Interactions ◽  
Somatic Hypermutation ◽  
Model Systems ◽  
Antigen Binding ◽  
Immunoglobulin Genes ◽  
Protein Protein Interactions ◽  
Cell Systems ◽  
High Affinity ◽  
Key Players

Somatic hypermutation (SHM) in immunoglobulin genes is required for high affinity antibody–antigen binding. Cultured cell systems, mouse model systems, and human genetic deficiencies have been the key players in identifying likely SHM pathways, whereas “pure” biochemical approaches have been far less prominent, but change appears imminent. Here we comment on how, when, and why biochemistry is likely to emerge from the shadows and into the spotlight to elucidate how the somatic mutation of antibody variable (V) regions is generated.

scholarly journals Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Avital Shushan ◽  
Mickey Kosloff
Keyword(s):  
Protein Interactions ◽  
Structural Elements ◽  
Protein Protein Interactions ◽  
Immunity Protein ◽  
Rational Engineering ◽  
High Affinity ◽  
Comparative Structure ◽  
Polar Interactions ◽  
Exquisite Specificity

AbstractThe interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein–protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1–α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1–α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein–protein interactions, with implications for drug development and rational engineering of these interfaces.

Following evolutionary paths to protein-protein interactions with high affinity and selectivity

2009 ◽  
Vol 16 (10) ◽  
pp. 1049-1055 ◽  
Author(s):  
Kalia Bernath Levin ◽  
Orly Dym ◽  
Shira Albeck ◽  
Shlomo Magdassi ◽  
Anthony H Keeble ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
High Affinity ◽  
Evolutionary Paths

scholarly journals The mechanistic GEMMs of oncogenic histones

2020 ◽  
Vol 29 (R2) ◽  
pp. R226-R235 ◽  
Author(s):  
Anders M Lindroth ◽  
Yoon Jung Park ◽  
Verónica Matía ◽  
Massimo Squatrito
Keyword(s):  
Protein Interactions ◽  
Drug Testing ◽  
Age Groups ◽  
Tumor Formation ◽  
Model Systems ◽  
Protein Protein Interactions ◽  
Efficient Treatment ◽  
Histone Modifiers ◽  
Second Tumors ◽  
The Stability

Abstract The last decade’s progress unraveling the mutational landscape of all age groups of cancer has uncovered mutations in histones as vital contributors of tumorigenesis. Here we review three new aspects of oncogenic histones: first, the identification of additional histone mutations potentially contributing to cancer formation; second, tumors expressing histone mutations to study the crosstalk of post-translational modifications, and; third, development of sophisticated biological model systems to reproduce tumorigenesis. At the outset, we recapitulate the firstly discovered histone mutations in pediatric and adolescent tumors of the brain and bone, which still remain the most pronounced histone alterations in cancer. We branch out to discuss the ramifications of histone mutations, including novel ones, that stem from altered protein-protein interactions of cognate histone modifiers as well as the stability of the nucleosome. We close by discussing animal models of oncogenic histones that reproduce tumor formation molecularly and morphologically and the prospect of utilizing them for drug testing, leading to efficient treatment and cure of deadly cancers with histone mutations.

Functional Display of Bioactive Peptides on the vGFP Scaffold.

2021 ◽  
Author(s):  
Sharon Min Qi Chee ◽  
Jantana Wongsantichon ◽  
Sze Yi Lau ◽  
Barindra Sana ◽  
Yuri Frosi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Bioactive Peptides ◽  
Negative Regulator ◽  
Live Cells ◽  
Structural Studies ◽  
Protein Protein Interactions ◽  
Target Interaction ◽  
High Affinity ◽  
Novel Scaffold

Abstract Grafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein-protein interactions.

Engineering High Affinity Protein–Protein Interactions Using a High-Throughput Microcapillary Array Platform

2016 ◽  
Vol 12 (2) ◽  
pp. 336-341 ◽  
Author(s):  
Sungwon Lim ◽  
Bob Chen ◽  
Mihalis S. Kariolis ◽  
Ivan K. Dimov ◽  
Thomas M. Baer ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
High Affinity ◽  
Array Platform

scholarly journals A Novel Method for Determination of the Affinity of Protein: Protein Interactions in Homogeneous Assays

2008 ◽  
Vol 13 (7) ◽  
pp. 674-682 ◽  
Author(s):  
Philip Newton ◽  
Paula Harrison ◽  
Stephen Clulow
Keyword(s):  
Protein Interactions ◽  
Competitive Inhibitor ◽  
Model Systems ◽  
Receptor Interaction ◽  
Affinity Constant ◽  
Protein Protein Interactions ◽  
Time Resolved ◽  
Protein Protein Interaction ◽  
Ox40 Ligand ◽  
Homogeneous Assays

Nonradioactive homogeneous assays are widely used to screen for inhibitors of biomolecular interactions. To ensure optimal sensitivity for the detection of competitive inhibitors, reagent concentrations should be fixed at or below the KD of the protein-protein interaction. Accurate measurement of KD during assay development is therefore critical. Although conventional methods work well with heterogeneous assays, they are generally unsatisfactory with homogeneous systems. Here the authors describe an alternative method to determine the KD of protein-protein interactions in homogeneous assays. The method uses a rearrangement of the Cheng-Prusoff equation: IC50= (([Ki]/KD) × [L]) + Ki. A competitive inhibitor is titrated into the ligand-receptor binding assay at a range of ligand concentrations and IC50 values are calculated. Plotting measured IC50 versus concentration of ligand gives a linear plot with y-intercept (Ki) and gradient (Ki/KD). KD is the affinity constant for the ligand-receptor interaction. Here the authors use homogeneous time-resolved fluorescence (HTRF®) in 2 model systems (TRAIL/TRAIL receptor 4 and OX40 ligand/OX40 receptor) and demonstrate that measured KD values calculated using the linearized Cheng-Prusoff plot compare favorably with those from independent experiments. The advantages and limitations of the method are discussed. ( Journal of Biomolecular Screening 2008:674-682)

scholarly journals Dependence of nucleotide substitutions on Ung2, Msh2, and PCNA-Ub during somatic hypermutation

2009 ◽  
Vol 206 (12) ◽  
pp. 2603-2611 ◽  
Author(s):  
Peter H.L. Krijger ◽  
Petra Langerak ◽  
Paul C.M. van den Berk ◽  
Heinz Jacobs
Keyword(s):  
B Cells ◽  
Proliferating Cell Nuclear Antigen ◽  
Mutation Frequency ◽  
Somatic Hypermutation ◽  
Nuclear Antigen ◽  
Immunoglobulin Genes ◽  
Nucleotide Substitutions ◽  
High Affinity ◽  
Proliferating Cell ◽  
Dependent Pathway

During somatic hypermutation (SHM), B cells introduce mutations into their immunoglobulin genes to generate high affinity antibodies. Current models suggest a separation in the generation of G/C transversions by the Ung2-dependent pathway and the generation of A/T mutations by the Msh2/ubiquitinated proliferating cell nuclear antigen (PCNA-Ub)–dependent pathway. It is currently unknown whether these pathways compete to initiate mutagenesis and whether PCNA-Ub functions downstream of Ung2. Furthermore, these models do not explain why mice lacking Msh2 have a more than twofold reduction in the total mutation frequency. Our data indicate that PCNA-Ub is required for A/T mutagenesis downstream of both Msh2 and Ung2. Furthermore, we provide evidence that both pathways are noncompetitive to initiate mutagenesis and even collaborate to generate half of all G/C transversions. These findings significantly add to our understanding of SHM and necessitate an update of present SHM models.

scholarly journals Functional display of bioactive peptides on the vGFP scaffold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sharon Min Qi Chee ◽  
Jantana Wongsantichon ◽  
Lau Sze Yi ◽  
Barindra Sana ◽  
Yuri Frosi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Bioactive Peptides ◽  
Negative Regulator ◽  
Live Cells ◽  
Structural Studies ◽  
Protein Protein Interactions ◽  
Target Interaction ◽  
High Affinity ◽  
Novel Scaffold

AbstractGrafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein–protein interactions.

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