Reactive Residues Research Articles

Highly efficient and freely soluble titanium sub-oxide powder as interfacial functional material for versatile photovoltaic cells

Metal suboxides (MOs) are among the most attractive interfacial materials owing to their outstanding functionality and excellent electrical and optical properties. However, the limitations of solution processability and ambient stability of the precursor hinder their widespread application. In this study, we developed a novel methodology for the solidification of sol–gel-derived MO-based organic complexes without affecting the properties of the MO products. By controlling the polarity of a conventional sol–gel derived titanium suboxide solution (STO) with organic functional side groups, we can easily obtain a nearly monodispersed solid-state precursor of STO (PTO) with excellent solubility in alcohols. PTO demonstrates ideal optical and electrical properties as an electron transport layer (ETL) with a significantly enhanced stability due to the elimination of the reactive residue. Owing to the optical, electrical, and excellent film-forming properties of PTO, organic and perovskite solar cells with PTO exhibit drastically improved performances and stability. Thus, a PTO-based ETL was successfully fabricated on various active layers, including those prepared from organic and perovskite materials, demonstrating the universality of PTO.

Improved process intermediate stability through the identification and elimination of reactive glycation residues – a monoclonal antibody case study

ABSTRACT The manufacturing of therapeutic biologics can result in a heterogeneous population of charge variants, encompassing many quality attributes which could impact activity and pharmacokinetics. Monitoring the relative abundance of these charge variants to demonstrate process consistency is an expectation of regulatory agencies. Control of the relative abundance of charge variants is also necessary to ensure product comparability across the product lifecycle. We have observed a significant shift in the relative abundance of charged species, as measured by capillary isoelectric focusing, during clarified cell culture fluid holds for several monoclonal antibodies. This lack of stability requires that the hold time for this process intermediate be significantly curtailed, eliminating manufacturing flexibility. We have identified the cause of this shift in relative abundance of charged species as changes in glycation levels, focused predominantly on three conserved, solvent accessible, lysine residues. Mutants of a model protein were generated that show increased charge state stability can be gained by eliminating these reactive lysines. Further, characterization studies were conducted on these mutants to determine the impact to biological activity and stability of the molecule, with no detrimental effects observed. Incorporating this knowledge into the assessments of candidate drugs could allow for the selection of molecules less susceptible to this product degradation pathway, allowing for greater manufacturing flexibility. This process of identifying and removing reactive lysine residues could be useful in the design of drug candidates with improved charge state stability, across a range of modalities.

The Integrated Stress Response and Eukaryotic Initiation Factor 2a Regulate Methylglyoxal-induced Pain Hypersensitivity

Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB. Diabetic neuropathic pain (DNP) is a debilitating condition. Current therapies are ineffective at treating DNP. We have previously demonstrated that methylglyoxal (MGO), a reactive metabolite of glycolysis that is elevated in the plasma of diabetic patients, pain hypersensitivity by engaging the integrated stress response (ISR) in mice. Induction of the ISR alters protein synthesis by inhibiting eukaryotic initiation factor 2α (eIF2α) and relying on eIF2A-mediated translation. We hypothesize that eIF2A regulates protein synthesis that contributes to pain hypersensitivity under conditions of ISR, especially after MGO application. We employed the recently characterized eIF2A-/- mouse to investigate how MGO treatment induces the ISR, facilitates eIF2A-mediated translation, and consequently causes pain hypersensitivity. We also subjected mice to tunicamycin (TUN), a well-known ISR inducer, and spared nerve injury (SNI). With donated human dorsal root ganglia (DRG) and spinal nerves (SpN), we aimed to translate our finding in rodents to humans. Intraplantar and intraperitoneal administrations of MGO induced pain hypersensitivity in a dose-dependent manner in male and female WT animals but not in eIF2A-/- mice. Following TUN and SNI, the eIF2A-/-¬¬ mice were protected against ISR-dependent pain (tunicamycin) but not against ISR-independent pain (SNI). MGO treatment of human DRG and SpN explants, and cultured human DRG neurons elevated p-eIF2α and eIF2A levels suggesting that MGO induces ISR not only in mice, but also in humans. Carboxyethyl lysine (CEL), a byproduct of reactive MGO and lysine residues, is increased in MGO treated cells and DRG and spinal nerve explants but not in tunicamycin treated explants, despite sharing elevated p-eIF2α and eIF2A levels. We demonstrate that eIF2A is necessary for the development of ISR-induced pain hypersensitivity. We further suggest that MGO and its byproduct, CEL, can be used as a biomarker to stratify DNP patients that would respond to ISR inhibitors like ISRIB.

Quantitative Analysis of Protein Bioconjugation in Cells with an Expanded Genetic Code

Protein bioconjugation – specific covalent bond formation between a protein of interest (POI) and a label – has a major impact on protein imaging, protein function studies, and drug discovery. Ideal protein bioconjugation should be fast, quantitative, one step with no off‐target binding, and have minimal impact on protein function, no matter the complexity of the biological environment. The impressive selectivity and kinetics of bioorthogonal reactions, specifically the catalyst‐free inverse‐electron demand Diels‐Alder (IEDDA) cycloaddition of tetrazines and strained alkene dienophiles, make IEDDA‐reactive unnatural amino acids an exceptional tool for protein bioconjugation via genetic code expansion. Analyses of these bioconjugation reactions in live cells has been mostly limited to fluorophore labeling, and thus is rarely quantitative. Self‐labeling enzymes, like HaloTag, have been used for protein isolation and purification, studies of protein‐protein interactions, and in vivo and in vitro imaging. The excellent permeability and low reactivity of the chloroalkane substrate of HaloTag yields bioconjugation detection independent of ligand permeability. The objective of this research is to utilize the HaloTag technology in combination with genetic code expansion to develop a generalized method for quantification of intracellular bioconjugation to any POI at defined sites. We hypothesize that bioconjugation efficiency could be assessed through site‐specific installation of a reactive handle on a POI, followed by ligand‐mediated protein labeling via ternary complex formation after cell lysis and exposure to HaloTag. Here we describe the optimization of concentrations, reaction times, and reaction partners for bioconjugation and pulldown for POIs expressing different IEDDA reactive surface residues. Additionally, we describe the attempted application of this method for labeling protein targets, such as kinases and nuclear receptors. With this new methodology, we have demonstrated quantitative assessment of protein bioconjugation reactions in live cells.

Donor-Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light.

Tryptophan (Trp) plays a variety of critical functional roles in protein biochemistry; however, owing to its low natural frequency and poor nucleophilicity, the design of effective methods for both single protein bioconjugation at Trp as well as for in situ chemoproteomic profiling remains a challenge. Here, we report a method for covalent Trp modification that is suitable for both scenarios by invoking photo-induced electron transfer (PET) as a means of driving efficient reactivity. We have engineered biaryl N-carbamoyl pyridinium salts that possess a donor-acceptor relationship that enables optical triggering with visible light whilst simultaneously attenuating the probe's photo-oxidation potential in order to prevent photodegradation. This probe was assayed against a small bank of eight peptides and proteins, where it was found that micromolar concentrations of the probe and short irradiation times (10-60 min) with violet light enabled efficient reactivity toward surface exposed Trp residues. The carbamate transferring group can be used to transfer useful functional groups to proteins including affinity tags and click handles. DFT calculations and other mechanistic analyses reveal correlations between excited state lifetimes, relative fluorescence quantum yields, and chemical reactivity. Biotinylated and azide-functionalized pyridinium salts were used for Trp profiling in HEK293T lysates and in situ in HEK293T cells using 440 nm LED irradiation. Peptide-level enrichment from live cell labeling experiments identified 290 Trp modifications, with 82% selectivity for Trp modification over other π-amino acids, demonstrating the ability of this method to identify and quantify reactive Trp residues from live cells.

Direct Intracellular Delivery of Benzene Diazonium Ions As Observed by Increased Tyrosine Phosphorylation.

A challenge within the field of bioconjugation is developing probes to uncover novel information on proteins and other biomolecules. Intracellular delivery of these probes offers the promise of giving relevant context to this information, and these probes can serve as hypothesis-generating tools within complex systems. Leveraging the utility of triazabutadiene chemistry, herein, we discuss the development of a probe that undergoes reduction-mediated deprotection to rapidly deliver a benzene diazonium ion (BDI) into cells. The intracellular BDI resulted in an increase in global tyrosine phosphorylation levels. Seeing phosphatase dysregulation as a potential source of this increase, a tyrosine phosphatase (PTP1B) was tested and shown to be both inhibited and covalently modified by the BDI. In addition to the expected azobenzene formation at tyrosine side chains, key reactive histidine residues were also modified.

A Review of Alpha-1 Antitrypsin Binding Partners for Immune Regulation and Potential Therapeutic Application.

Alpha-1 antitrypsin (AAT) is the canonical serine protease inhibitor of neutrophil-derived proteases and can modulate innate immune mechanisms through its anti-inflammatory activities mediated by a broad spectrum of protein, cytokine, and cell surface interactions. AAT contains a reactive methionine residue that is critical for its protease-specific binding capacity, whereby AAT entraps the protease on cleavage of its reactive centre loop, neutralises its activity by key changes in its tertiary structure, and permits removal of the AAT-protease complex from the circulation. Recently, however, the immunomodulatory role of AAT has come increasingly to the fore with several prominent studies focused on lipid or protein-protein interactions that are predominantly mediated through electrostatic, glycan, or hydrophobic potential binding sites. The aim of this review was to investigate the spectrum of AAT molecular interactions, with newer studies supporting a potential therapeutic paradigm for AAT augmentation therapy in disorders in which a chronic immune response is strongly linked.

Cysteine Reactivity Profiling to Unveil Redox Regulation in Phytopathogens.

Reactivity-based chemical proteomics is a powerful technology based on the use of tagged chemicals that covalently react with surface-exposed residues on proteins in native proteomes. Reactivity profiling involves the purification, identification, and quantification of labeled peptides by LC-MS/MS. Here, we have detailed a protocol for reactivity profiling of Cys residues using iodoacetamide probes, displaying >1000 reactive Cys residues in the proteome of phytopathogen Pseudomonas syringae pv. tomato DC3000 (PtoDC3000). Comparative reactivity profiling of PtoDC3000 treated with or without hydrogen peroxide (H2O2) identified ~200 H2O2-sensitive Cys residues in antioxidant enzymes, metabolic enzymes, and transcription regulators. Interestingly, half of these H2O2-sensitive Cys residues are more reactive in response to H2O2 and several proteins have multiple Cys residues with opposite reactivities in response to H2O2 exposure.

Redox proteomic study of Bacillus cereus thiol proteome during fermentative anaerobic growth

BackgroundBacillus cereus is a notorious foodborne pathogen, which can grow under anoxic conditions. Anoxic growth is supported by endogenous redox metabolism, for which the thiol redox proteome serves as an interface. Here, we studied the cysteine (Cys) proteome dynamics of B. cereus ATCC 14579 cells grown under fermentative anoxic conditions. We used a quantitative thiol trapping method combined with proteomics profiling.ResultsIn total, we identified 153 reactive Cys residues in 117 proteins participating in various cellular processes and metabolic pathways, including translation, carbohydrate metabolism, and stress response. Of these reactive Cys, 72 were detected as reduced Cys. The B. cereus Cys proteome evolved during growth both in terms of the number of reduced Cys and the Cys-containing proteins identified, reflecting its growth-phase-dependence. Interestingly, the reduced status of the B. cereus thiol proteome increased during growth, concomitantly to the decrease of extracellular oxidoreduction potential.ConclusionsTaken together, our data show that the B. cereus Cys proteome during unstressed fermentative anaerobic growth is a dynamic entity and provide an important foundation for future redox proteomic studies in B. cereus and other organisms.

Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Post-translational Modifications of the Protein Termini.

Post-translational modifications (PTM) involve enzyme-mediated covalent addition of functional groups to proteins during or after synthesis. These modifications greatly increase biological complexity and are responsible for orders of magnitude change between the variety of proteins encoded in the genome and the variety of their biological functions. Many of these modifications occur at the protein termini, which contain reactive amino- and carboxy-groups of the polypeptide chain and often are pre-primed through the actions of cellular machinery to expose highly reactive residues. Such modifications have been known for decades, but only a few of them have been functionally characterized. The vast majority of eukaryotic proteins are N- and C-terminally modified by acetylation, arginylation, tyrosination, lipidation, and many others. Post-translational modifications of the protein termini have been linked to different normal and disease-related processes and constitute a rapidly emerging area of biological regulation. Here we highlight recent progress in our understanding of post-translational modifications of the protein termini and outline the role that these modifications play in vivo.

Reactive and non-reactive residue curve maps analysis to produce Butyl Lactate by catalytic distillation

This work presents a novel methodology that combines the scattered information in the literature for the construction of residue curve maps through a global optimization technique. The synthesis of Butyl Lactate from Lactic Acid and 1-Butanol was considered as case study since this compound is increasingly used as green solvent, flavor in the food industry, and in pharmaceutical formulations. Initially, a review on reported experimental thermodynamic data for the system involved in the Butyl Lactate synthesis was done. Then, a validation of binary parameters was performed according to the NRTL activity model, which replicates properly the available information. The kinetic model of the system was validated, and the reactive curve maps were calculated at different Damköhler (Da) values. The model predicts different azeotropes at 5 and 101.325 kPa and the systems were topologically consistent according to the thermodynamic evaluation. Based on the analysis of residue curve maps, Butyl Lactate can be obtained in the bottom stream. Finally, a conceptual design to produce Butyl Lactate by reactive distillation was proposed by using the generated residue maps.

Photosensitized Oxidative Dimerization at Tyrosine by a Water-Soluble 4-Amino-1,8-naphthalimide.

The oxidation of proteins generates reactive amino acid (AA) residue intermediates, leading to protein modification and cross-linking. Aerobic studies with peptides and photosensitizers allow for the controlled generation of reactive oxygen species (ROS) and reactive AA residue intermediates, providing mechanistic insights as to how natural protein modifications form. Such studies have inspired the development of abiotic methods for protein modification and crosslinking, including applications of biomedical importance. Dityrosine linkages derived from oxidation at tyrosine (Tyr) residues represent one of the more well-understood oxidation-induced modifications. Here we demonstrate an aerobic, visible light-dependent oxidation reaction of Tyr-containing substrates promoted by a water-soluble 4-amino-1,8-naphthalimide-based photosensitizer. The developed procedure converts Tyr-containing substrates into o,o'-Tyr-Tyr linked dimers. The regioselectively formed o,o'-Tyr-Tyr linkage is consistent with dimeric standards prepared using a known enzymatic method. A crossover study with two peptides provides a statistical mixture of three distinct o,o'-Tyr-Tyr linked dimers, supporting a mechanism that involves Tyr residue oxidation followed by intermolecular combination.

Investigation into the reaction of reactive dyes with carboxylate salts and the application of carboxylate‐modified reactive dyes to cotton

AbstractInk‐jet printing of cellulosic fabrics with reactive dyes typically requires that the fabric is pretreated with alkali, prior to printing, to facilitate efficient fixation of the dye. In this paper we evaluate the use of sodium formate and other carboxylate salts as a neutral (pH 6.5) pretreatment process. The thickened, prepared‐for‐print pad liquor contained at least 50 gdm−3 of the selected carboxylate salt and was applied to the cotton fabrics by a pad‐dry procedure. The fabric was then ink‐jet printed with reactive dye inks, followed by standard steaming and washing‐off processes. The pH of the carboxylate salt pretreatment was 6.5 and the aqueous extracts from the print fabrics at the end of the steaming process remained at pH 6.5. It was observed that even at pH 6.5, in the presence of selected carboxylates, significant reactive dye fixation could be achieved on a cotton substrate, whereas in the absence of the carboxylate, very little or even zero fixation was achieved. Infrared and capillary electrophoresis analyses of model reactions of reactive dyes with the carboxylate salts indicated that reactive ester residues were formed, and which subsequently promoted reaction with the cellulosic substrates. In addition to improving reactive dye fixation in ink‐jet printing, the carboxylate‐modified dyes were also demonstrated to improve long‐liquor dyeing properties on cotton substrates. As an extension of this carboxylate‐based printing process, the incorporation of lithium acetate (100 gdm−3) into the ink formulation was further studied and it was demonstrated that the necessity for a preparative pretreatment process could be eliminated.

Glycation of albumin and its implication in Diabetes: A comprehensive analysis using mass spectrometry

AimTo understand the mechanism of glycation of albumin and effects on cysteinylation and methionine oxidation. MethodsThe in vitro glycation of HSA and BSA was studied with varying concentrations of glucose. Clinical blood samples of diabetic subjects with varying HbA1c values, were analyzed to assess in vivo glycation. All samples and their tryptic digests were analyzed using liquid chromatography/mass spectrometry. Glycation sites were mapped on to the three-dimensional structure of the HSA and BSA. ResultsA total thirty-one sites for glycation and eight sites of Nε-carboxymethyl-lysine (CML) modification were identified on albumin. The site selectivity of glycation was correlated with the environment of the reactive residue in the three-dimensional structure. ConclusionsThe maximum percentage glycation under extreme conditions was in the range of ~55 to 88% in four weeks. Two major glycation sites K-233 and K-525 were identified, which together accounted for 40–50% of total glycation. A correlation was observed between glycation and oxidation of methionine residues in samples glycated in vitro. The role of spatially proximate residues in facilitating the glycation process is evident. The tri- and tetra-glycated isoforms of albumin can serve as biomarkers for the severe uncontrolled diabetic state.

New insights into the cholesterol esterase- and lipase-inhibiting potential of bioactive peptides from camel whey hydrolysates: Identification, characterization, and molecular interaction

Novel antihypercholesterolemic bioactive peptides (BAP) from peptic camel whey protein hydrolysates (CWPH) were generated at different time, temperature, and enzyme concentration (%). Hydrolysates showed higher pancreatic lipase- (PL; except 3 CWPH) and cholesterol esterase (CE)-inhibiting potential, as depicted by lower half-maximal inhibitory concentration values (IC50 values) compared with nonhydrolyzed camel whey proteins (CWP). Peptide sequencing and in silico data depicted that most BAP from CWPH could bind active site of PL, whereas as only 3 peptides could bind the active site of CE. Based on higher number of reactive residues in the BAP and greater number of substrate binding sites, FCCLGPVPP was identified as a potential CE-inhibitory peptide, and PAGNFLPPVAAAPVM, MLPLMLPFTMGY, and LRFPL were identified as PL inhibitors. Molecular docking of selected peptides showed hydrophilic and hydrophobic interactions between peptides and target enzymes. Thus, peptides derived from CWPH warrant further investigation as potential candidates for adjunct therapy for hypercholesterolemia.

The number of catalytic cycles in an enzyme’s lifetime and why it matters to metabolic engineering

Metabolic engineering uses enzymes as parts to build biosystems for specified tasks. Although a part's working life and failure modes are key engineering performance indicators, this is not yet so in metabolic engineering because it is not known how long enzymes remain functional in vivo or whether cumulative deterioration (wear-out), sudden random failure, or other causes drive replacement. Consequently, enzymes cannot be engineered to extend life and cut the high energy costs of replacement. Guided by catalyst engineering, we adopted catalytic cycles until replacement (CCR) as a metric for enzyme functional life span in vivo. CCR is the number of catalytic cycles that an enzyme mediates in vivo before failure or replacement, i.e., metabolic flux rate/protein turnover rate. We used estimated fluxes and measured protein turnover rates to calculate CCRs for ∼100-200 enzymes each from Lactococcus lactis, yeast, and Arabidopsis CCRs in these organisms had similar ranges (<103 to >107) but different median values (3-4 × 104 in L. lactis and yeast versus 4 × 105 in Arabidopsis). In all organisms, enzymes whose substrates, products, or mechanisms can attack reactive amino acid residues had significantly lower median CCR values than other enzymes. Taken with literature on mechanism-based inactivation, the latter finding supports the proposal that 1) random active-site damage by reaction chemistry is an important cause of enzyme failure, and 2) reactive noncatalytic residues in the active-site region are likely contributors to damage susceptibility. Enzyme engineering to raise CCRs and lower replacement costs may thus be both beneficial and feasible.

Time-Resolved Acetaldehyde-Based Accessibility Profiling Maps Ligand-Target Interactions.

Elucidating ligand-protein interactions is important in understanding the biochemical machinery for given proteins. Previously, formaldehyde (FH)-based labeling has been employed to obtain such structural knowledge, since reactive residues that participate in ligand-target interactions display reduced accessibility to FH-labeling reagents, and thus can be identified by quantitative proteomics. Although being rapid and efficient for probing proteinaceous lysine accessibility, here, we report an acetaldehyde (AcH)-labeling approach that complements with FH for probing ligand-target interactions. AcH labeling examines lysine accessibility at a more moderate reaction speed and hence delivers a cleaner reaction when compared to that of FH. The subsequent application of AcH to label RNase A without and with ligands has assisted to assign lysines involved in ligand-RNase A binding by detecting the time-dependent changes in accessibility profiles. We further employed multiple reaction monitoring (MRM) to quantify these ligand-binding-responsive sites when a variety of potential ligands were queried. We noted that the time-resolved abundance changes of these peptides can sensitively determine the ligand-binding sites and differentiate binding affinities among these ligands, which was confirmed by native mass spectrometry (MS) and molecular docking. Lastly, we demonstrated that the binding sites can be recognized by monitoring the chemical accessibility of these responsive peptides in cell lysates. Together, we believe that the proposed combined use of AcH-based lysine accessibility profiling, native MS, and MRM screening is a powerful toolbox in characterizing ligand-target interactions, mapping topography, and interrogating affinities and holds promise for future applications in a complex cellular environment.

Methylmercury-Mediated Oxidative Stress and Activation of the Cellular Protective System.

Methylmercury (MeHg) is a well-known neurotoxicant that causes severe intoxication in humans. In Japan, it is referred to as Minamata disease, which involves two characteristic clinical forms: fetal type and adult type depending on the exposed age. In addition to MeHg burden level, individual susceptibility to MeHg plays a role in the manifestation of MeHg toxicity. Research progress has pointed out the importance of oxidative stress in the pathogenesis of MeHg toxicity. MeHg has a high affinity for selenohydryl groups, sulfhydryl groups, and selenides. It has been clarified that such affinity characteristics cause the impairment of antioxidant enzymes and proteins, resulting in the disruption of antioxidant systems. Furthermore, MeHg-induced intracellular selenium deficiency due to the greater affinity of MeHg for selenohydryl groups and selenides leads to failure in the recoding of a UGA codon for selenocysteine and results in the degradation of antioxidant selenoenzyme mRNA by nonsense-mediated mRNA decay. The defect of antioxidant selenoenzyme replenishment exacerbates MeHg-mediated oxidative stress. On the other hand, it has also been revealed that MeHg can directly activate the antioxidant Keap1/Nrf2 signaling pathway. This review summarizes the incidence of MeHg-mediated oxidative stress from the viewpoint of the individual intracellular redox system interactions and the MeHg-mediated aforementioned intracellular events. In addition, the mechanisms of cellular stress pathways and neuronal cell death triggered by MeHg-mediated oxidative stress and direct interactions of MeHg with reactive residues of proteins are mentioned.

Tyrosine Conjugation Methods for Protein Labelling.

Over the last two decades, the development of chemical biology and the need for more defined protein conjugates have fostered active research on new bioconjugation techniques. In particular, a wide range of biorthogonal labelling strategies have been reported to functionalise the phenol side chain of tyrosines (Tyr). Tyr occur at medium frequency and are partially buried at the protein surface, offering interesting opportunities for site-selective labelling of the most reactive residues. Tyr-targeting has proved effective for designing a wide range of important biomolecules including antibody-drug conjugates, fluorescent or radioactive protein probes, glycovaccines, protein aggregates, and PEG conjugates. Innovative methods have also been reported for site-specific labelling with ligand-directed anchors and for the specific affinity capture of proteins. This review will present and discuss these promising alternatives to the conventional labelling of the nucleophilic lysine and cysteine residues.