Cysteine Residues In Proteins Research Articles

Post-Translational Modifications to Cysteine Residues in Plant Proteins and Their Impact on the Regulation of Metabolism and Signal Transduction.

Cys is one of the least abundant amino acids in proteins. However, it is often highly conserved and is usually found in important structural and functional regions of proteins. Its unique chemical properties allow it to undergo several post-translational modifications, many of which are mediated by reactive oxygen, nitrogen, sulfur, or carbonyl species. Thus, in addition to their role in catalysis, protein stability, and metal binding, Cys residues are crucial for the redox regulation of metabolism and signal transduction. In this review, we discuss Cys post-translational modifications (PTMs) and their role in plant metabolism and signal transduction. These modifications include the oxidation of the thiol group (S-sulfenylation, S-sulfinylation and S-sulfonylation), the formation of disulfide bridges, S-glutathionylation, persulfidation, S-cyanylation S-nitrosation, S-carbonylation, S-acylation, prenylation, CoAlation, and the formation of thiohemiacetal. For each of these PTMs, we discuss the origin of the modifier, the mechanisms involved in PTM, and their reversibility. Examples of the involvement of Cys PTMs in the modulation of protein structure, function, stability, and localization are presented to highlight their importance in the regulation of plant metabolic and signaling pathways.

S-Nitrosylation of p39 promotes its degradation and contributes to synaptic dysfunction induced by β-amyloid peptide

Alzheimer’s disease (AD), characterized by cognitive decline, is increasingly recognized as a disorder marked by synaptic loss and dysfunction. Despite this understanding, the underlying pathophysiological mechanisms contributing to synaptic impairment remain largely unknown. In this study, we elucidate a previously undiscovered signaling pathway wherein the S-nitrosylation of the Cdk5 activator p39, a post-translational modification involving the addition of nitric oxide to protein cysteine residues, plays a crucial role in synaptic dysfunction associated with AD. Our investigation reveals heightened p39 S-nitrosylation in the brain of an amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model of AD. Additionally, soluble amyloid-β oligomers (Aβ), implicated in synaptic loss in AD, induce p39 S-nitrosylation in cultured neurons. Notably, we uncover that p39 protein level is regulated by S-nitrosylation, with nitric oxide S-nitrosylating p39 at Cys265 and subsequently promoting its degradation. Furthermore, our study demonstrates that S-nitrosylation of p39 at Cys265 significantly contributes to amyloid-β (Aβ) peptide-induced dendrite retraction and spine loss. Collectively, our findings highlight S-nitrosylation of p39 as a novel aberrant redox protein modification involved in the pathogenesis of AD, suggesting its potential as a therapeutic target for the disease.

Photoinduced Electron-Transfer-Mediated Differential Recognition of Proteins on Plasmonic Surfaces.

Photoinduced enhanced Raman spectroscopy (PIERS) has emerged as an efficient technique for enhancing the vibrational modes of analyte molecules adsorbed on a plasmonic nanoparticle-semiconductor hybrid material through chemical enhancement governed by electron transfer from the semiconductor to the plasmonic nanoparticles under an additional ultraviolet (UV) preirradiation step. The increase in chemical enhancement is imperative in analyzing and detecting pharmaceutically important moieties, such as amino acids and proteins, with a low Raman scattering cross section, even in complex biological environments. Herein, we demonstrate that UV preirradiation induced the creation of additional oxygen vacancies by introducing a low concentration (≈1%) of Ni as a dopant in the 2D platelike morphology of the BiOCl semiconductor; i.e., defect states in the semiconductor can induce charge transfer from the semiconductor to the plasmonic nanoparticles. This phenomenon facilitates electron transfer to the adsorbed analyte on the plasmonic surface. Additionally, we have shown the usefulness of this method in protein immobilization on the substrate surface, followed by the identification of a specific protein in the mixture of proteins. Proteins containing cysteine residues capture these electrons to form a surface-bound thiol group via a transient disulfide electron adduct radical. This allows differential binding of the protein molecules to the semiconductor plasmonic hybrid depending on the concentration of surface cysteine residues in proteins. Through PIERS and principal component analysis, we demonstrate the possibility of probing and distinguishing biomolecules based on their surface composition and secondary structure components even in their mixtures, thus paving the way for efficient analysis of complex biological systems.

The importance of stratum corneum ω-linoleoyloxyacylceramides in human skin barrier health: their biochemistry, processing enzymes and metabolites involved in corneocyte lipid envelope maturation.

Over the past 50 years there have been great strides made in the discovery of the composition and relevance of the total stratum corneum (SC) ceramide matrix. However, the focus of this review is on the free intercellular class of ω-linoleoyloxyacylceramides, corneocyte-bound ceramides and associated lipids known as the corneocyte lipid envelope (CLE) together with their processing enzymes involved in aiding ceramide attachment the corneocyte protein envelope (CPE). Two structural models and partially shared biosynthetic pathways have been proposed for the attachment of CPE-bound O-ceramides (ω-hydroxyceramides attached to glutamate residues of proteins in the (CPE) using the 12R-lipoxygenase (12R-LOX)/epidermal lipoxygenase-3 (eLOX3)/epoxide hydrolase-3 (EPHX3)/unknown esterase/ transglutaminase-1 (TG1) attachment pathway) and CPE-bound EO-ceramides (epoxy-enone ceramides attached to cysteine residues of proteins in the CPE using the 12R-LOX/eLOX3/short chain dehydrogenase/reductase family 9C member 7 (SDR9C7)/non-enzymatic attachment pathway), i.e. there is a bifurcation step beyond epidermal eLOX3. Their formation and structures will be discussed as well as their relevance in compromised skin barrier conditions together with our own work on SC maturation examined by proteomics, lipidomics, enzyme immunolocalization studies, mechanical fragility assays and Nile red staining of corneocyte envelopes (CE). Reduced levels of 12R-LOX, eLOX3, SDR9C7 and TG1 were observed in photodamaged skin of the cheeks that were associated with reduced SC maturation as evidenced by Nile red staining and increased CE fragility. In the severely photodamaged cheeks of Albino African SC we also observed increased levels of acylceramides. Concomitantly by reducing the activity of 12R-LOX by antibody inhibition and TG1 inhibition with a known chemical inhibitor, we demonstrated in a humidity-based exvivo SC maturation model that these enzymes contributed to increased CE hydrophobicity and mechanical integrity. We hypothesize that at least the CPE-bound O-ceramide pathway is operational in the SC. Nevertheless, our understanding of the full complexity of ω-linoleoyloxyacylceramides and the composition of the CLE is limited particularly on cosmetically relevant body sites such as the face.

Mechanism of Methylene Blue Inducing the Disulfide Bond Formation of Tubulin-Associated Unit Proteins.

Methylene blue (MB) has recently completed a Phase-3 clinical trial as leuco-methylthioninium (LMT) bis(hydromethanesulfonate) for treating Alzheimer's disease. Herein, we investigated the mechanism underlying the MB inhibition of tubulin-associated unit (tau) aggregation by focusing on tau monomers. We found that MB causes disulfide bond formation, resulting in strong nuclear magnetic resonance chemical shift perturbations in a large area of tau proteins. The oxidized form of MB, namely methylthioninium (MT+), specifically catalyzed the oxidation of cysteine residues in tau proteins to form disulfide bonds directly using O2. This process is independent of the MT+-to-LMT redox cycle. Moreover, MT+ preferentially oxidized C291 and C322 in the lysine-rich R2 and R3 domains. Under in vivo brain physoxia conditions, LMT may convert to MT+, possibly interfering with tau fibrillation via disulfide bond formation.

Glutathione Induces Keap1 S-Glutathionylation and Mitigates Oscillating Glucose-Induced β-Cell Dysfunction by Activating Nrf2.

Glutathione (GSH), a robust endogenous antioxidant, actively participates in the modulation of the redox status of cysteine residues in proteins. Previous studies have indicated that GSH can prevent β-cell failure and prediabetes caused by chronic oscillating glucose (OsG) administration. However, the precise mechanism underlying the protective effect is not well understood. Our current research reveals that GSH is capable of reversing the reduction in Nrf2 levels, as well as downstream genes Grx1 and HO-1, in the islet β-cells of rats induced by chronic OsG. In vitro experiments have further demonstrated that GSH can prevent β-cell dedifferentiation, apoptosis, and impaired insulin secretion caused by OsG. Additionally, GSH facilitates the translocation of Nrf2 into the nucleus, resulting in an upregulation of Nrf2-targeted genes such as GCLC, Grx1, HO-1, and NQO1. Notably, when the Nrf2 inhibitor ML385 is employed, the effects of GSH on OsG-treated β-cells are abrogated. Moreover, GSH enhances the S-glutathionylation of Keap1 at Cys273 and Cys288, but not Cys151, in OsG-treated β-cells, leading to the dissociation of Nrf2 from Keap1 and facilitating Nrf2 nuclear translocation. In conclusion, the protective role of GSH against OsG-induced β-cell failure can be partially attributed to its capacity to enhance Keap1 S-glutathionylation, thereby activating the Nrf2 signaling pathway. These findings provide novel insights into the prevention and treatment of β-cell failure in the context of prediabetes/diabetes, highlighting the potential of GSH.

S-nitrosylation-triggered unfolded protein response maintains hematopoietic progenitors in Drosophila

The Drosophila lymph gland houses blood progenitors that give rise to myeloid-like blood cells. Initially, blood progenitors proliferate, but later, they become quiescent to maintain multipotency before differentiation. Despite the identification of various factors involved in multipotency maintenance, the cellular mechanism controlling blood progenitor quiescence remains elusive. Here, we identify the expression of nitric oxide synthase in blood progenitors, generating nitric oxide for post-translational S-nitrosylation of protein cysteine residues. S-nitrosylation activates the Ire1-Xbp1-mediated unfolded protein response, leading to G2 cell-cyclearrest. Specifically, we identify the epidermal growth factor receptor as a target of S-nitrosylation, resulting in its retention within the endoplasmic reticulum and blockade of its receptor function. Overall, our findings highlight developmentally programmed S-nitrosylation as a critical mechanism that induces protein quality control in blood progenitors, maintaining their undifferentiated state by inhibiting cell-cycle progression and rendering them unresponsive to paracrine factors.

Insights into the Multifaceted Roles of Thioredoxin-1 System: Exploring Knockout Murine Models.

Redox balance is increasingly identified as a major player in cellular signaling. A fundamentally simple reaction of oxidation and reduction of cysteine residues in cellular proteins is the central concept in this complex regulatory mode of protein function. Oxidation of key cysteine residues occurs at the physiological levels of reactive oxygen species (ROS), but they are reduced by a supply of thiol antioxidant molecules including glutathione, glutaredoxin, and thioredoxin. While these molecules show complex compensatory roles in experimental conditions, transgenic animal models provide a comprehensive picture to pinpoint the role of each antioxidant. In this review, we have specifically focused on the available literature on thioredoxin-1 system transgenic models that include thioredoxin and thioredoxin reductase proteins. As the identification of thioredoxin protein targets is technically challenging, the true contribution of this system in maintaining cellular balance remains unidentified, including the role of this system in the brain.

Protein S-palmitoylation modification: implications in tumor and tumor immune microenvironment.

Protein S-palmitoylation is a reversible post-translational lipid modification that involves the addition of a 16-carbon palmitoyl group to a protein cysteine residue via a thioester linkage. This modification plays a crucial role in the regulation protein localization, accumulation, secretion, stability, and function. Dysregulation of protein S-palmitoylation can disrupt cellular pathways and contribute to the development of various diseases, particularly cancers. Aberrant S-palmitoylation has been extensively studied and proven to be involved in tumor initiation and growth, metastasis, and apoptosis. In addition, emerging evidence suggests that protein S-palmitoylation may also have a potential role in immune modulation. Therefore, a comprehensive understanding of the regulatory mechanisms of S-palmitoylation in tumor cells and the tumor immune microenvironment is essential to improve our understanding of this process. In this review, we summarize the recent progress of S-palmitoylation in tumors and the tumor immune microenvironment, focusing on the S-palmitoylation modification of various proteins. Furthermore, we propose new ideas for immunotherapeutic strategies through S-palmitoylation intervention.

Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized.NEW & NOTEWORTHY Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.

Hydrogen Sulfide (H2S)/Polysulfides (H2Sn) Signalling and TRPA1 Channels Modification on Sulfur Metabolism.

Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2) produced by enzymes play a role as signalling molecules regulating neurotransmission, vascular tone, cytoprotection, inflammation, oxygen sensing, and energy formation. H2Sn, which have additional sulfur atoms to H2S, and other S-sulfurated molecules such as cysteine persulfide and S-sulfurated cysteine residues of proteins, are produced by enzymes including 3-mercaptopyruvate sulfurtransferase (3MST). H2Sn are also generated by the chemical interaction of H2S with NO, or to a lesser extent with H2O2. S-sulfuration (S-sulfhydration) has been proposed as a mode of action of H2S and H2Sn to regulate the activity of target molecules. Recently, we found that H2S/H2S2 regulate the release of neurotransmitters, such as GABA, glutamate, and D-serine, a co-agonist of N-methyl-D-aspartate (NMDA) receptors. H2S facilitates the induction of hippocampal long-term potentiation, a synaptic model of memory formation, by enhancing the activity of NMDA receptors, while H2S2 achieves this by activating transient receptor potential ankyrin 1 (TRPA1) channels in astrocytes, potentially leading to the activation of nearby neurons. The recent findings show the other aspects of TRPA1 channels-that is, the regulation of the levels of sulfur-containing molecules and their metabolizing enzymes. Disturbance of the signalling by H2S/H2Sn has been demonstrated to be involved in various diseases, including cognitive and psychiatric diseases. The physiological and pathophysiological roles of these molecules will be discussed.

Evaluation of the Drug-Induced Liver Injury Potential of Saxagliptin through Reactive Metabolite Identification in Rats.

A liver injury was recently reported for saxagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor. However, the underlying mechanisms of saxagliptin-induced liver injury remain unknown. This study aimed to evaluate whether saxagliptin, a potent and selective DPP-4 inhibitor that is globally used for treating type 2 diabetes mellitus, binds to the nucleophiles in vitro. Four DPP-4 inhibitors, including vildagliptin, were evaluated for comparison. Only saxagliptin and vildagliptin, which both contain a cyanopyrrolidine group, quickly reacted with L-cysteine to enzyme-independently produce thiazolinic acid metabolites. This saxagliptin-cysteine adduct was also found in saxagliptin-administered male Sprague-Dawley rats. In addition, this study newly identified cysteinyl glycine conjugates of saxagliptin and 5-hydroxysaxagliptin. The observed metabolic pathways were hydroxylation and conjugation with cysteine, glutathione, sulfate, and glucuronide. In summary, we determined four new thiazoline-containing thiol metabolites (cysteine and cysteinylglycine conjugates of saxagliptin and 5-hydroxysaxagliptin) in saxagliptin-administered male rats. Our results reveal that saxagliptin can covalently bind to the thiol groups of cysteine residues of endogenous proteins in vivo, indicating the potential for saxagliptin to cause drug-induced liver injury.

A Prognostic Activity of Glutaredoxin 1 Protein (Grx1) in Colon Cancer.

Glutaredoxin 1 (Grx1) is an essential enzyme that regulates redox signal transduction and repairs protein oxidation by reversing S-glutathionylation, an oxidative modification of protein cysteine residues. Grx1 removes glutathione from proteins to restore their reduced state (protein-SH) and regulate protein-SSG levels in redox signaling networks. Thus, it can exert an influence on the development of cancer. To further investigate this problem, we performed an analysis of Grx1 expression in colon adenocarcinoma samples from the Polish population of patients with primary colon adenocarcinoma (stages I and II of colon cancer) and those with regional lymph node metastasis (stage III of colon cancer). Our study revealed a significant correlation between the expression of Grx1 protein through immunohistochemical analysis and various clinical characteristics of patients, such as histological grade, depth of invasion, angioinvasion, staging, regional lymph node invasion, and PCNA expression. It was found that almost 88% of patients with stage I had high levels of Grx1 expression, while only 1% of patients with stage III exhibited high levels of Grx1 protein expression. Furthermore, the study discovered that high levels of Grx1 expression were present in samples of colon mucosa without any pathological changes. These results were supported by in vitro analysis conducted on colorectal cancer cell lines that corresponded to stages I, II, and III of colorectal cancer, using qRT-PCR and Western blot.

Methods to monitor mitochondrial disulfide bonds.

Mitochondria contain numerous proteins that utilize the chemistry of cysteine residues, which can be reversibly oxidized. These proteins are involved in mitochondrial biogenesis, protection against oxidative stress, metabolism, energy transduction to adenosine triphosphate, signaling and cell death among other functions. Many proteins located in the mitochondrial intermembrane space are imported by the mitochondrial import and assembly pathway the activity of which is based on the reversible oxidation of cysteine residues and oxidative trapping of substrates. Oxidative modifications of cysteine residues are particularly difficult to study because of their labile character. Here we present techniques that allow for monitoring the oxidative state of mitochondrial proteins as well as to investigate the mitochondrial import and assembly pathway. This chapter conveys basic concepts on sample preparation and techniques to monitor the redox state of cysteine residues in mitochondrial proteins as well as the strategies to study mitochondrial import and assembly pathway.

Evidence that protein thiols are not primary targets of intracellular reactive oxygen species in growing Escherichia coli.

The oxidizability of cysteine residues is exploited in redox chemistry and as a source of stabilizing disulfide bonds, but it also raises the possibility that these side chains will be oxidized when they should not be. It has often been suggested that intracellular oxidative stress from hydrogen peroxide or superoxide may result in the oxidation of the cysteine residues of cytoplasmic proteins. That view seemed to be supported by the discovery that one cellular response to hydrogen peroxide is the induction of glutaredoxin 1 and thioredoxin 2. In this study we used model compounds as well as alkaline phosphatase to test this idea. Our results indicate that molecular oxygen, superoxide, and hydrogen peroxide are very poor oxidants of N-acetylcysteine and of the protein thiols of alkaline phosphatase in vitro. Copper could accelerate thiol oxidation, but iron did not. When alkaline phosphatase was engineered to remain in the cytoplasm of live cells, unnaturally high concentrations of hydrogen peroxide were required to oxidize it to its active, disulfide-dependent form, and toxic levels of superoxide had no effect. At the same time, far lower concentrations of these oxidants were sufficient to poison key metalloenzymes. The elimination of glutaredoxin 1 and thioredoxin 2 did not change these results, raising the question of why E. coli induces them during peroxide stress. In fact, when catalase/peroxidase mutants were chronically stressed with hydrogen peroxide, the absence of glutaredoxin 1 and thioredoxin 2 did not impair growth at all, even in a minimal medium over many generations. We conclude that physiological levels of reduced oxygen species are not potent oxidants of typical protein thiols. Glutaredoxin and thioredoxin must either have an alternative purpose or else play a role under culture conditions that differ from the ones we tested.

Sulfur signaling pathway in cardiovascular disease

Hydrogen sulfide (H2S) and sulfur dioxide (SO2), recognized as endogenous sulfur-containing gas signaling molecules, were the third and fourth molecules to be identified subsequent to nitric oxide and carbon monoxide (CO), and exerted diverse biological effects on the cardiovascular system. However, the exact mechanisms underlying the actions of H2S and SO2 have remained elusive until now. Recently, novel post-translational modifications known as S-sulfhydration and S-sulfenylation, induced by H2S and SO2 respectively, have been proposed. These modifications involve the chemical alteration of specific cysteine residues in target proteins through S-sulfhydration and S-sulfenylation, respectively. H2S induced S-sulfhydrylation can have a significant impact on various cellular processes such as cell survival, apoptosis, cell proliferation, metabolism, mitochondrial function, endoplasmic reticulum stress, vasodilation, anti-inflammatory response and oxidative stress in the cardiovascular system. Alternatively, S-sulfenylation caused by SO2 serves primarily to maintain vascular homeostasis. Additional research is warranted to explore the physiological function of proteins with specific cysteine sites, despite the considerable advancements in comprehending the role of H2S-induced S-sulfhydration and SO2-induced S-sulfenylation in the cardiovascular system. The primary objective of this review is to present a comprehensive examination of the function and potential mechanism of S-sulfhydration and S-sulfenylation in the cardiovascular system. Proteins that undergo S-sulfhydration and S-sulfenylation may serve as promising targets for therapeutic intervention and drug development in the cardiovascular system. This could potentially expedite the future development and utilization of drugs related to H2S and SO2.

Lysinoalanine cross-linking is a conserved post-translational modification in the spirochete flagellar hook.

Spirochetes cause Lyme disease, leptospirosis, syphilis, and several other human illnesses. Unlike other bacteria, spirochete flagella are enclosed within the periplasmic space where the filaments distort and push the cell body by the action of the flagellar motors. We previously demonstrated that the oral pathogen Treponema denticola (Td) and Lyme disease pathogen Borreliella burgdorferi (Bb) form covalent lysinoalanine (Lal) cross-links between conserved cysteine and lysine residues of the FlgE protein that composes the flagellar hook. In Td, Lal is unnecessary for hook assembly but is required for motility, presumably due to the stabilizing effect of the cross-link. Herein, we extend these findings to other, representative spirochete species across the phylum. We confirm the presence of Lal cross-linked peptides in recombinant and in vivo-derived samples from Treponema spp., Borreliella spp., Brachyspira spp., and Leptospira spp. As was observed with Td, a mutant strain of Bb unable to form the cross-link has greatly impaired motility. FlgE from Leptospira spp. does not conserve the Lal-forming cysteine residue which is instead substituted by serine. Nevertheless, Leptospira interrogans FlgE also forms Lal, with several different Lal isoforms being detected between Ser-179 and Lys-145, Lys-148, and Lys-166, thereby highlighting species or order-specific differences within the phylum. Our data reveal that the Lal cross-link is a conserved and necessary posttranslational modification across the spirochete phylum and may thus represent an effective target for the development of spirochete-specific antimicrobials.

Desulfurative Borylation of Small Molecules, Peptides, and Proteins.

We introduce a direct conversion of alkyl thiols into boronic acids, facilitated by a water-soluble phosphine, 1,3,5-triaza-7-phosphaadamantane (PTA), in conjunction with tetrahydroxydiboron (B2(OH)4), acting as both a radical initiator and a boron source. This desulfurative borylation reaction has been successfully applied to various substrates, including cysteine residues in oligopeptides and small proteins, primary alkyl thiols found in pharmaceutical compounds, disulfides, and selenocysteine. Optimization of reaction conditions was undertaken to reduce the formation of unwanted reactions, such as the reduction of alanyl or other primary radicals, and to prevent deleterious reactions between the phosphine and N-terminal amine that lead to methylene adducts by utilizing a buffer containing glycine-glycine (GG) dipeptide. The developed method is characterized by its operational simplicity and robustness. Moreover, its compatibility with various functional groups present in peptides and proteins makes it a promising tool for late-stage functionalization, extending its potential application across a broad spectrum of chemical and biological targets.

H2O2-dependent oxidation of the transcription factor GmNTL1 promotes salt tolerance in soybean.

Reactive oxygen species (ROS) play an essential role in plant growth and responses to environmental stresses. Plant cells sense and transduce ROS signaling directly via hydrogen peroxide (H2O2)-mediated posttranslational modifications (PTMs) on protein cysteine residues. Here, we show that the H2O2-mediated cysteine oxidation of NAC WITH TRANS-MEMBRANE MOTIF1-LIKE 1 (GmNTL1) in soybean (Glycine max) during salt stress promotes its release from the endoplasmic reticulum (ER) membrane and translocation to the nucleus. We further show that an oxidative posttranslational modification on GmNTL1 residue Cys-247 steers downstream amplification of ROS production by binding to and activating the promoters of RESPIRATORY BURST OXIDASE HOMOLOG B (GmRbohB) genes, thereby creating a feed-forward loop to fine-tune GmNTL1 activity. In addition, oxidation of GmNTL1 Cys-247 directly promotes the expression of CATION H+ EXCHANGER 1 (GmCHX1)/SALT TOLERANCE-ASSOCIATED GENE ON CHROMOSOME 3 (GmSALT3) and Na+/H+ Antiporter 1 (GmNHX1). Accordingly, transgenic overexpression of GmNTL1 in soybean increases the H2O2 levels and K+/Na+ ratio in the cell, promotes salt tolerance, and increases yield under salt stress, while an RNA interference-mediated knockdown of GmNTL1 elicits the opposite effects. Our results reveal that the salt-induced oxidation of GmNTL1 promotes its relocation and transcriptional activity through an H2O2-mediated posttranslational modification on cysteine that improves resilience of soybean against salt stress.

Effects of spike proteins on angiotensin converting enzyme 2 (ACE2)

The Coronavirus Disease 2019 (COVID-19) pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enters host cells through interactions of its spike protein to Angiotensin-Converting Enzyme 2 (ACE2). ACE2 is a peptidase that cleaves Angiotensin II, a critical pathological mediator. This study investigated if the spike protein binding to ACE2 compromises its peptidase activity. Spike/ACE2 Binding Assays suggested that spike proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, but not HKU1, bind to ACE2. S1 and receptor-binding domain (RBD), but not S2, extracellular domain (ECD) or CendR domain, bind to ACE2. While glycosylated spike proteins prepared in HEK293 cells bind to ACE2, non-glycosylated proteins produced in E. coli do not. Cysteine residues of the spike protein expressed in HEK293 cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of HEK cell-produced protein attenuates the ACE2 binding, while the oxidation of the E. coli protein does not promote the binding. The S1 protein of SARS-CoV-2 enhances the ACE2 peptidase activity, while SARS-CoV, MERS-CoV or HKU1 does not. The ACE2 activity is enhanced by RBD, but not ECD or CendR. In contrast to distinct ACE2 binding capacities of proteins expressed in HEK293 cells and in E. coli, spike proteins expressed in both systems enhance the ACE2 activity. Thus, the spike protein of SARS-CoV-2, but not other coronaviruses, enhances the ACE2 peptidase activity through its RBD in a glycosylation-independent manner.