scholarly journals Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

2017 ◽  
Vol 114 (4) ◽  
pp. E486-E495 ◽  
Author(s):  
Patrick Koenig ◽  
Chingwei V. Lee ◽  
Benjamin T. Walters ◽  
Vasantharajan Janakiraman ◽  
Jeremy Stinson ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Conformational Dynamics ◽  
Human Antibody ◽  
Antigen Binding ◽  
Mutational Landscape ◽  
Antibody Maturation ◽  
Binding Function ◽  
Variable Domains

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

scholarly journals Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties

2019 ◽  
Vol 117 (1) ◽  
pp. 292-299 ◽  
Author(s):  
Lynn E. Macdonald ◽  
Karoline A. Meagher ◽  
Matthew C. Franklin ◽  
Natasha Levenkova ◽  
Johanna Hansen ◽  
...  
Keyword(s):  
Small Molecules ◽  
Binding Pocket ◽  
Human Antibody ◽  
Antigen Binding ◽  
Wild Type ◽  
Antigen Binding Site ◽  
Binding Properties ◽  
Variable Domains ◽  
Constant Domains ◽  
Ig Heavy Chain

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

Structural Requirements of the Major Protective Antibody to Haemophilus influenzae Type b

1999 ◽  
Vol 67 (5) ◽  
pp. 2503-2514 ◽  
Author(s):  
Lotte Hougs ◽  
Lars Juul ◽  
Arne Svejgaard ◽  
Torben Barington
Keyword(s):  
Haemophilus Influenzae ◽  
Somatic Mutations ◽  
Affinity Maturation ◽  
Dimensional Structure ◽  
Haemophilus Influenzae Type ◽  
Antigen Binding ◽  
Immunoglobulin Gene ◽  
Type B

ABSTRACT Protective antibodies to the important childhood pathogenHaemophilus influenzae type b (Hib) are directed against the capsular polysaccharide (HibCP). Most of the antibody is encoded by a well-defined set of (“canonical”) immunoglobulin genes, including the Vκ A2 gene, and expresses an idiotypic marker (HibId-1). In comparison to noncanonical antibodies, the canonical antibody is generally of higher avidity, shows higher levels of in vitro bactericidal activity, and is more protective in infant rats. Using site-directed mutagenesis, we here characterize canonical HibCP antibodies expressed as antigen-binding fragments (Fabs) in Escherichia coli, define amino acids involved in antigen binding and idiotype expression, and propose a three-dimensional structure for the variable domains. We found that canonical Fabs, unlike a noncanonical Fab, bound effectively to HibCP in the absence of somatic mutations. Nevertheless, pronounced mutation-based affinity maturation was demonstrated in vivo. An almost perfect correlation was found between unmutated gene segments that mediated binding in vitro and those encoding canonical HibCP antibodies in vivo. Thus, the Vκ A2a gene could be replaced by the A2c gene but not by the highly homologous sister gene, A18b, corresponding to the demonstrated usage of A2c but not of A18b in vivo. Similarly, only Jκ1 and Jκ3, which predominate in the response in vivo, were able to facilitate binding in vitro. These findings suggest that the restricted immunoglobulin gene usage in HibCP antibodies reflects strict structural demands ensuring relatively high affinity prior to somatic mutations—requirements met by only a limited spectrum of immunoglobulin gene combinations.

scholarly journals Emerging Role of Long Non-Coding RNAs in the Pathobiology of Glioblastoma

2021 ◽  
Vol 10 ◽  
Author(s):  
Omidvar Rezaei ◽  
Kasra Honarmand Tamizkar ◽  
Guive Sharifi ◽  
Mohammad Taheri ◽  
Soudeh Ghafouri-Fard
Keyword(s):  
Somatic Mutations ◽  
Invasion And Metastasis ◽  
Poor Survival ◽  
Proliferation And Apoptosis ◽  
Non Coding Rnas ◽  
The Impact ◽  
Therapeutic Responses

Glioblastoma is the utmost aggressive diffuse kind of glioma which is originated from astrocytes, neural stem cells or progenitors. This malignant tumor has a poor survival rate. A number of genetic aberrations and somatic mutations have been associated with this kind of cancer. In recent times, the impact of long non-coding RNAs (lncRNAs) in glioblastoma has been underscored by several investigations. Up-regulation of a number of oncogenic lncRNAs such as H19, MALAT1, SNHGs, MIAT, UCA, HIF1A-AS2 and XIST in addition to down-regulation of other tumor suppressor lncRNAs namely GAS5, RNCR3 and NBAT1 indicate the role of these lncRNAs in the pathogenesis of glioblastoma. Several in vitro and a number of in vivo studies have demonstrated the contribution of these transcripts in the regulation of cell proliferation and apoptosis, cell survival, invasion and metastasis of glioblastoma cells. Moreover, some lncRNAs such as SBF2-AS1 are involved in conferring resistance to temozolomide. Finally, few circularRNAs have been identified that influence the evolution of glioblastoma. In this paper, we discuss the impacts of lncRNAs in the pathogenesis of glioblastoma, their applications as markers and their implications in the therapeutic responses in this kind of cancer.

scholarly journals Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

2020 ◽  
Vol 295 (28) ◽  
pp. 9583-9595
Author(s):  
Aleksei Agapov ◽  
Artem Ignatov ◽  
Matti Turtola ◽  
Georgiy Belogurov ◽  
Daria Esyunina ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Stress Responses ◽  
Active Site ◽  
Rna Synthesis ◽  
Conformational Dynamics ◽  
Dna Lesions ◽  
Trigger Loop ◽  
Nucleotide Addition

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins, in vitro transcription, kinetic analyses, and in vivo cell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis by Escherichia coli RNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxic in vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival.

Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study

1990 ◽  
Vol 48 (2) ◽  
pp. 166-167
Author(s):  
W.A. Jacob ◽  
R. Hertsens ◽  
A. Van Bogaert ◽  
M. De Smet
Keyword(s):  
Energy Metabolism ◽  
Calcium Ions ◽  
Energy Production ◽  
Regulation Of Respiration ◽  
Free Calcium ◽  
Mitochondrial Matrix ◽  
Cytochemical Study ◽  
Nmr Techniques

In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Potential Uses of Vinegar as a Medicine and Related in vivo Mechanisms

2016 ◽  
Vol 86 (3-4) ◽  
pp. 127-151 ◽  
Author(s):  
Zeshan Ali ◽  
Zhenbin Wang ◽  
Rai Muhammad Amir ◽  
Shoaib Younas ◽  
Asif Wali ◽  
...  
Keyword(s):  
Chemical Structure ◽  
Review Paper ◽  
Heart Diseases ◽  
Folk Medicine ◽  
Active Role ◽  
Current Review ◽  
Different Types ◽  
Positive Effect

While the use of vinegar to fi ght against infections and other crucial conditions dates back to Hippocrates, recent research has found that vinegar consumption has a positive effect on biomarkers for diabetes, cancer, and heart diseases. Different types of vinegar have been used in the world during different time periods. Vinegar is produced by a fermentation process. Foods with a high content of carbohydrates are a good source of vinegar. Review of the results of different studies performed on vinegar components reveals that the daily use of these components has a healthy impact on the physiological and chemical structure of the human body. During the era of Hippocrates, people used vinegar as a medicine to treat wounds, which means that vinegar is one of the ancient foods used as folk medicine. The purpose of the current review paper is to provide a detailed summary of the outcome of previous studies emphasizing the role of vinegar in treatment of different diseases both in acute and chronic conditions, its in vivo mechanism and the active role of different bacteria.

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