Tyrosine Modifications in Aging

This is the publisher's version, also available electronically from http://online.liebertpub.com/doi/abs/10.1089/ars.2012.4595Significance: The understanding of physiological and pathological processes involving protein oxidation, particularly under conditions of aging and oxidative stress, can be aided by proteomic identification of proteins that accumulate oxidative post-translational modifications only if these detected modifications are connected to functional consequences. The modification of tyrosine (Tyr) residues can elicit significant changes in protein structure and function, which, in some cases, may contribute to biological aging and age-related pathologies, such as atherosclerosis, neurodegeneration, and cataracts. Recent Advances: Studies characterizing proteins in which Tyr has been modified to 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, 3,3′-dityrosine and other cross-links, or 3-chlorotyrosine are reviewed, with an emphasis on structural and functional consequences. Critical Issues: Distinguishing between inconsequential modifications and functionally significant ones requires careful biochemical and biophysical analysis of target proteins, as well as innovative methods for isolating the effects of the multiple modifications that often occur under oxidizing conditions. Future Directions: The labor-intensive task of isolating and characterizing individual modified proteins must continue, especially given the expanding list of known modifications. Emerging approaches, such as genetic and metabolic incorporation of unnatural amino acids, hold promise for additional focused studies of this kind. Antioxid. Redox Signal. 17, 1571–1579

Exploring oxidative modifications of tyrosine:an update on mechanisms of formation, advances in analysis and biological consequences

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology

Chemical Stability of the Botanical Drug Substance Crofelemer: A Model System for Comparative Characterization of Complex Mixture Drugs

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.As the second of a 3-part series of articles in this issue concerning the development of a mathematical model for comparative characterization of complex mixture drugs using crofelemer (CF) as a model compound, this work focuses on the evaluation of the chemical stability profile of CF. CF is a biopolymer containing a mixture of proanthocyanidin oligomers which are primarily composed of gallocatechin with a small contribution from catechin. CF extracted from drug product was subjected to molecular weight–based fractionation and thiolysis. Temperature stress and metal-catalyzed oxidation were selected for accelerated and forced degradation studies. Stressed CF samples were size fractionated, thiolyzed, and analyzed with a combination of negative-ion electrospray ionization mass spectrometry (ESI-MS) and reversed-phase-HPLC with UV absorption and fluorescence detection. We further analyzed the chemical stability data sets for various CF samples generated from reversed-phase-HPLC-UV and ESI-MS using data-mining and machine learning approaches. In particular, calculations based on mutual information of over 800,000 data points in the ESI-MS analytical data set revealed specific CF cleavage and degradation products that were differentially generated under specific storage/degradation conditions, which were not initially identified using traditional analysis of the ESI-MS results

Comparative Characterization of Crofelemer Samples Using Data Mining and Machine Learning Approaches With Analytical Stability Data Sets

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.There is growing interest in generating physicochemical and biological analytical data sets to compare complex mixture drugs, for example, products from different manufacturers. In this work, we compare various crofelemer samples prepared from a single lot by filtration with varying molecular weight cutoffs combined with incubation for different times at different temperatures. The 2 preceding articles describe experimental data sets generated from analytical characterization of fractionated and degraded crofelemer samples. In this work, we use data mining techniques such as principal component analysis and mutual information scores to help visualize the data and determine discriminatory regions within these large data sets. The mutual information score identifies chemical signatures that differentiate crofelemer samples. These signatures, in many cases, would likely be missed by traditional data analysis tools. We also found that supervised learning classifiers robustly discriminate samples with around 99% classification accuracy, indicating that mathematical models of these physicochemical data sets are capable of identifying even subtle differences in crofelemer samples. Data mining and machine learning techniques can thus identify fingerprint-type attributes of complex mixture drugs that may be used for comparative characterization of products

Intramolecular hydrogen transfer reactions of thiyl radicals from glutathione: formation of carbon-centered radical at Glu, Cys and Gly

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3000494Glutathione thiyl radicals (GS•) were generated in H2O and D2O by either exposure of GSH to AAPH#, photoirradiation of GSH in the presence of acetone, or photoirradiation of GSSG. Detailed interpretation of the fragmentation pathways of deuterated GSH and GSH-derivatives during mass spectrometry analysis allowed us to demonstrate that reversible intramolecular H-atom transfer reactions between GS• and C-H bonds at Cys[αC], Cys[βC], and Gly[αC] are possible

The Storage and In-Use Stability of mRNA Vaccines and Therapeutics: Not A Cold Case

The remarkable impact of mRNA vaccines on mitigating disease and improving public health has been amply demonstrated during the COVID-19 pandemic. Many new mRNA-based vaccine and therapeutic candidates are in development, yet the current reality of their stability limitations requires their frozen storage. Numerous challenges remain to improve formulated mRNA stability and enable refrigerator storage, and this review provides an update on developments to tackle this multi-faceted stability challenge. We describe the chemistry underlying mRNA degradation during storage and highlight how lipid nanoparticle (LNP) formulations are a double-edged sword: while LNPs protect mRNA against enzymatic degradation, interactions with and between LNP excipients introduce additional risks for mRNA degradation. We also discuss strategies to improve mRNA stability both as a drug substance (DS) and a drug product (DP) including the (1) design of the mRNA molecule (nucleotide selection, primary and secondary structures), (2) physical state of the mRNA-LNP complexes, (3) formulation composition and purity of the components, and (4) DS and DP manufacturing processes. Finally, we summarize analytical control strategies to monitor and assure the stability of mRNA-based candidates, and advocate for an integrated analytical and formulation development approach to further improve their storage, transport, and in-use stability profiles

Oxidized and Aggregated Recombinant Human Interferon Beta is Immunogenic in Human Interferon Beta Transgenic Mice

PurposeTo study the effect of oxidation on the structure of recombinant human interferon beta-1a (rhIFNβ-1a) and its immunogenicity in wild-type and immune-tolerant transgenic mice.MethodsUntreated rhIFNβ-1a was degraded by metal-catalyzed oxidation, H2O2-mediated oxidation, and guanidine-mediated unfolding/refolding. Four rhIFNβ-1a preparations with different levels of oxidation and aggregation were injected intraperitoneally in mice 15× during 3 weeks. Both binding and neutralizing antibodies were measured.ResultsAll rhIFNβ-1a preparations contained substantial amounts of aggregates. Metal-catalyzed oxidized rhIFNβ-1a contained high levels of covalent aggregates as compared with untreated rhIFNβ-1a. H2O2-treated rhIFNβ-1a showed an increase in oligomer and unrecovered protein content by HP-SEC; RP-HPLC revealed protein oxidation. Guanidine-treated rhIFNβ-1a mostly consisted of dimers and oligomers and some non-covalent aggregates smaller in size than those in untreated rhIFNβ-1a. All degraded samples showed alterations in tertiary protein structure. Wild-type mice showed equally high antibody responses against all preparations. Transgenic mice were discriminative, showing elevated antibody responses against both metal-catalyzed oxidized and H2O2-treated rhIFNβ-1a as compared to untreated and guanidine-treated rhIFNβ-1a.ConclusionsOxidation-mediated aggregation increased the immunogenicity of rhIFNβ-1a in transgenic mice, whereas aggregated preparations devoid of measurable oxidation levels were hardly immunogenic

The Interaction of Hypotaurine and Other Sulfinates with Reactive Oxygen and Nitrogen Species:A Survey of Reaction Mechanisms

Considerable strides have been made in understanding the oxidative mechanisms involved in the final steps of the cysteine pathway leading to taurine. The oxidation of sulfinates, hypotaurine and cysteine sulfinic acid, to the respective sulfonates, taurine and cysteic acid, has never been associated with any specific enzyme. Conversely, there is strong evidence that in vivo formation of taurine and cysteic acid is the result of sulfinate interaction with a variety of biologically relevant oxidants. In the last decade, many experiments have been performed to understand whether peroxynitrite, nitrogen dioxide and carbonate radical anion could be included in the biologically relevant reactive species capable of oxidizing sulfinates. Thanks to this work, it has been possible to highlight two possible reaction mechanisms (direct and indirect reaction) of sulfinates with reactive oxygen and nitrogen species.The sulfinates oxidation, mediated by peroxynitrite, is an example of both reaction mechanisms: through a two-electron-direct-reaction with peroxynitrite or through a one-electron-indirect-transfer reaction. In the indirect mechanism, the peroxynitrite homolysis releases hydroxyl and nitrogen dioxide radical and in addition the degradation of short-lived adduct formed by peroxynitrite and CO2 can generate carbonate radical anion. The reaction of hypotaurine and cysteine sulfinic acid with peroxynitrite-derived radicals is accompanied by extensive oxygen uptake with the generation of transient intermediates, which can begin a reaction by an oxygen-dependent mechanism with the sulfonates, taurine, and cysteic acid as final products. Due to pulse radiolysis studies, it has been shown that transient sulfonyl radicals (RSO2(•)) have been produced during the oxidation of both sulfinates by one-electron transfer reaction.The purpose is to analyze all the aspects of the reactive mechanism in the sulfinic group oxidation of hypotaurine and cysteine sulfinic acid through the results obtained from our laboratory in recent years

Hyperacute Directional Hearing and Phonotactic Steering in the Cricket (Gryllus bimaculatus deGeer)

Background: Auditory mate or prey localisation is central to the lifestyle of many animals and requires precise directional hearing. However, when the incident angle of sound approaches 0u azimuth, interaural time and intensity differences gradually vanish. This poses a demanding challenge to animals especially when interaural distances are small. To cope with these limitations imposed by the laws of acoustics, crickets employ a frequency tuned peripheral hearing system. Although this enhances auditory directionality the actual precision of directional hearing and phonotactic steering has never been studied in the behaviourally important frontal range. Principal Findings: Here we analysed the directionality of phonotaxis in female crickets (Gryllus bimaculatus) walking on an open-loop trackball system by measuring their steering accuracy towards male calling song presented at frontal angles of incidence. Within the range of 630u, females reliably discriminated the side of acoustic stimulation, even when the sound source deviated by only 1u from the animal’s length axis. Moreover, for angles of sound incidence between 1u and 6u the females precisely walked towards the sound source. Measuring the tympanic membrane oscillations of the front leg ears with a laser vibrometer revealed between 0u and 30u a linear increasing function of interaural amplitude differences with a slope of 0.4 dB/u. Auditory nerve recordings closely reflected these bilateral differences in afferent response latency and intensity that provide the physiological basis for precise auditory steering