Cysteine Oxidation Research Articles

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.

CysQuant: Simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

Oxidation is an underappreciated post-translational modification in the regulation of immune responses associated with changes in phosphorylation.

Although macrophages are known to be affected by their redox status, oxidation is not yet a well-recognized post-translational modification (PTM) in regulating macrophages and immune cells in general. While it has been described that the redox status of single cysteines in specific proteins is relevant for macrophage functions, global oxidation information is scarce. Hence, we globally assessed the impact of oxidation on macrophage activation using untargeted proteomics and PTM-omics. We exposed THP-1 macrophages to lipopolysaccharide (LPS) for 4 h and 24 h and applied a sequential iodoTMT labeling approach to get information on overall oxidation as well as reversible oxidation of cysteines. Thus, we identified 10452 oxidation sites, which were integratively analyzed with 5057 proteins and 7148 phosphorylation sites to investigate their co-occurance with other omics layers. Based on this integrative analysis, we found significant upregulation of several immune-related pathways, e.g. toll-like receptor 4 (TLR4) signaling, for which 19 proteins, 7 phosphorylation sites, and 39 oxidation sites were significantly affected, highlighting the relevance of oxidations in TLR4-induced macrophage activation. Co-regulation of oxidation and phosphorylation was observed, as evidenced by multiply modified proteins related to inflammatory pathways. Additionally, we observed time-dependent effects, with differences in the dynamics of oxidation sites compared to proteins and phosphorylation sites. Overall, this study highlights the importance of oxidation in regulating inflammatory processes and provides a method that can be readily applied to study the cellular redoxome globally.

Ahp deficiency-induced redox imbalance leads to metabolic alterations in E.coli

Alkyl hydroperoxide reductase (Ahp) is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli (E. coli). Ahp-deficient strains have been found to have high reactive oxygen species (ROS) levels, sufficient to cause cell damage. However, the exact role and underlying mechanisms of Ahp deficiency-induced cell damage remain largely unknown. Here, the E. coli MG1655 ΔAhp mutant strain was constructed as a model of deficiency to assess its role. The cells of the ΔAhp strain were found to be significantly longer than those of the wild strain, with elevated ROS and hydrogen peroxide (H2O2) levels. Proteome, redox proteome and metabolome analyses were performed to systematically present a global and quantitative profile and delineate the redox signaling and metabolic alterations at the proteome, metabolome, and cysteine oxidation site levels. The multiomics data revealed that Ahp deficiency disrupted the redox balance, activated the OxyR system, upregulated oxidative defense proteins and inhibited the TCA cycle to some extent. Surprisingly, the mutant strain shifted from aerobic respiration to anaerobic respiration and fermentation during the logarithmic phase in the presence of sufficient O2. The acid resistance system was activated to mitigate the effect of excessive acid produced by fermentation. Taken together, the results of this study demonstrated that Ahp deficiency triggered cellular redox imbalance and regulated metabolic pathways to confer resistance to submicromolar intracellular H2O2 levels in E. coli.

PL02.1.A DELINEATING AND TARGETING A NOVEL METABOLISM-BASED POST-TRANSLATIONAL MECHANISM REGULATING THE ABUNDANCE OF THE ‘UNDRUGGABLE’ ONCOPROTEIN C-MYC IN MEDULLOBLASTOMA

Abstract BACKGROUND Brain tumors are the leading cause of cancer death in children, and medulloblastoma (MB) is one of the most common pediatric central nervous system malignancies. Amplification of the c-MYC oncogene is frequently observed in the most aggressive and lethal subgroup of this disease, group 3 (G3), but not in other subgroups. Patients that have G3 MB tumors with high c-MYC abundance are more likely to present as metastatic and are prone to develop fatal recurrent tumors. Unfortunately, the functional ubiquity and disordered structure of c-MYC makes it difficult to target for cancer treatment. Therefore, it is critical to identify novel, out-of-the-box strategies to suppress oncogenic c-MYC in highly aggressive G3 MB brain tumors. Recently, metabolism has emerged as a major regulator of overall cellular signaling processes through post-translational and epigenetic mechanisms. While c-MYC is known to regulate cellular metabolism, whether metabolism plays a role in reciprocally supporting enhanced c-MYC abundance in cancer is unknown. We hypothesize that an intrinsic feedback mechanism may exist where metabolic activity modulates c-MYC abundance that could be exploited as a therapeutic strategy to improve outcomes for G3 MB patients. MATERIAL AND METHODS Using various well-characterized G3 MB cells, patient-derived G3 MB cells, orthotopic intracerebellar xenograft models, patient tumor bioinformatics, detailed biochemical characterization, and point-mutation analyses, we have identified a novel metabolism-dependent post-translational modification that regulates c-MYC stability in G3 MB. RESULTS In-depth molecular analyses unveiled that c-MYC is susceptible to oxidation and proteasomal degradation under conditions of metabolic stress. Targeting mitochondrial respiration via inhibition of complex-I promotes the accumulation of reactive oxygen species (ROS) and leads to rapid cysteine oxidation and proteasomal degradation of c-MYC in G3 MB cells, but not normal human brain astrocytes or neural stem cells (NSCs). Point mutation analysis combined with biochemical oxidation assays identified the specific cysteine residues of c-MYC that are susceptible to oxidation and ultimately responsible for enhanced c-MYC degradation following complex-I inhibition. Importantly, oral administration of a blood-brain barrier permeable complex-I inhibitor impaired the growth of intracerebellar MB xenograft tumors in mice, significantly prolonging animal survival. CONCLUSION Altogether, these findings unveil a novel mechanism through which metabolism regulates the post-translational stability of c-MYC and provides insights for designing rationale therapeutic strategies for the treatment of MB patients. SUPPORT This work is supported by a project grant from the Canadian Institutes of Health Research (CIHR).

THE ROLE OF SULFUR-CONTAINING AMINO ACIDS IN THE PREVENTIVE NUTRITION OF WORKERS IN CONTACT WITH CHROMIUM

In the world, a large number of workers are exposed to fumes, mists and dusts containing Chromium and its compounds. One of the ways to prevent chromium intoxication is the enrichment of food rations with substances that have antioxidant and complexing ability. Attention is focused on the recommendations on the use of sulfur-containing amino acids and sulfur-rich proteins in the composition of food products for the prevention of intoxication in workers who come into contact with chromium in production. The mechanism of reduction of hexavalent chromium compounds by cysteine was considered. It has been shown that the process of reduction of chromates with cysteine is accompanied by the oxidation of cysteine to cystine and subsequent complexation with trivalent chromium. The structure of chromium III cystinate was confirmed by IR and electron spectroscopy and elemental analysis. It has been established that in the formed chelate complex [Cr(SNO2C3H6)6Cr], chromium (III) has a bond with cystine through the amino and carboxyl groups, LD50 is 2100 mg/kg and is less toxic than inorganic chromium (III) salts. The article analyzes protein raw materials rich in cysteine, which can be recommended to satisfy the daily requirement of cysteine, or to reduce chromium intoxication of workers working in a work area with a high content of chromium salts.

Performance benchmarking microplate-immunoassays for quantifying target-specific cysteine oxidation reveals their potential for understanding redox-regulation and oxidative stress.

The antibody-linked oxi-state assay (ALISA) for quantifying target-specific cysteine oxidation can benefit specialist and non-specialist users. Specialists can benefit from time-efficient analysis and high-throughput target and/or sample n-plex capacities. The simple and accessible "off-the-shelf" nature of ALISA brings the benefits of oxidative damage assays to non-specialists studying redox-regulation. Until performance benchmarking establishes confidence in the "unseen" microplate results, ALISA is unlikely to be widely adopted. Here, we implemented pre-set pass/fail criteria to benchmark ALISA by robustly evaluating immunoassay performance in diverse biological contexts. ELISA-mode ALISA assays were accurate, reliable, and sensitive. For example, the average inter-assay CV for detecting 20%- and 40%-oxidised PRDX2 or GAPDH standards was 4.6% (range: 3.6-7.4%). ALISA displayed target-specificity. Immunodepleting the target decreased the signal by ∼75%. Single-antibody formatted ALISA failed to quantify the matrix-facing alpha subunit of the mitochondrial ATP synthase. However, RedoxiFluor quantified the alpha subunit displaying exceptional performance in the single-antibody format. ALISA discovered that (1) monocyte-to-macrophage differentiation amplified PRDX2-specific cysteine oxidation in THP-1cells and (2) exercise increased GAPDH-specific cysteine oxidation in human erythrocytes. The "unseen" microplate data were "seen-to-be-believed" via orthogonal visually displayed immunoassays like the dimer method. Finally, we established target (n=3) and sample (n=100) n-plex capacities in ∼4h with 50-70min hands-on time. Our work showcases the potential of ALISA to advance our understanding of redox-regulation and oxidative stress.

Structure-function analysis suggests that the photoreceptor LITE-1 is a light-activated ion channel

Sensation of light is essential for all organisms. The eye-less nematode Caenorhabditis elegans detects UV and blue light to evoke escape behavior. The photosensor LITE-1 absorbs UV photons with an unusually high extinction coefficient, involving essential tryptophans. Here, we modeled the structure and dynamics of LITE-1 using AlphaFold2-multimer and molecular dynamics (MD) simulations and performed mutational and behavioral assays in C.elegans to characterize its function. LITE-1 resembles olfactory and gustatory receptors from insects, recently shown to be tetrameric ion channels. We identified residues required for channel gating, light absorption, and mechanisms of photo-oxidation, involving a likely binding site for the peroxiredoxin PRDX-2. Furthermore, we identified the binding pocket for a putative chromophore. Several residues lining this pocket have previously been established as essential for LITE-1 function. A newly identified critical cysteine pointing into the pocket represents a likely chromophore attachment site. We derived a model for how photon absorption, via a network of tryptophans and other aromatic amino acids, induces an excited state that is transferred to the chromophore. This evokes conformational changes in the protein, possibly leading to a state receptive to oxidation of cysteines and, jointly, to channel gating. Electrophysiological data support the idea that LITE-1 is a photon and H2O2-coincidence detector. Other proteins with similarity to LITE-1, specifically C.elegans GUR-3, likely use a similar mechanism for photon detection. Thus, a common protein fold and assembly, used for chemoreception in insects, possibly by binding of a particular compound, may have evolved into a light-activated ion channel.

Grafting Strategies of Oxidation-Prone Coiled-Coil Peptides for Protein Capture in Bioassays: Impact of Orientation and the Oxidation State.

Many biomedical and biosensing applications require functionalization of surfaces with proteins. To this end, the E/K coiled-coil peptide heterodimeric system has been shown to be advantageous. First, Kcoil peptides are covalently grafted onto a given surface. Ecoil-tagged proteins can then be non-covalently captured via a specific interaction with their Kcoil partners. Previously, oriented Kcoil grafting was achieved via thiol coupling, using a unique Kcoil with a terminal cysteine residue. However, cysteine-terminated Kcoil peptides are hard to produce, purify, and oxidize during storage. Indeed, they tend to homodimerize and form disulfide bonds via oxidation of their terminal thiol group, making it impossible to later graft them on thiol-reactive surfaces. Kcoil peptides also contain multiple free amine groups, available for covalent coupling through carbodiimide chemistry. Grafting Kcoil peptides on surfaces via amine coupling would thus guarantee their immobilization regardless of their terminal cysteine's oxidation state, at the expense of the control over their orientation. In this work, we compare Kcoil grafting strategies for the subsequent capture of Ecoil-tagged proteins, for applications such as surface plasmon resonance (SPR) biosensing and cell culture onto protein-decorated substrates. We compare the "classic" thiol coupling of cysteine-terminated Kcoil peptides to the amine coupling of (i) monomeric Kcoil and (ii) dimeric Kcoil-Kcoil linked by a disulfide bond. We have observed that SPR biosensing performances relying on captured Ecoil-tagged proteins were similar for amine-coupled dimeric Kcoil-Kcoil and thiol-coupled Kcoil peptides, at the expense of higher Ecoil-tagged protein consumption. For cell culture applications, Ecoil-tagged growth factors captured on amine-coupled monomeric Kcoil signaled through cell receptors similarly to those captured on thiol-coupled Kcoil peptides. Altogether, while oriented thiol coupling of cysteine-terminated Kcoil peptides remains the most reliable and versatile platform for Ecoil-tagged protein capture, amine coupling of Kcoil peptides, either monomeric or dimerized through a cysteine bond, can offer a good alternative when the challenges and costs associated with the production of monomeric cysteine-tagged Kcoil are too dissuasive for the application.

Mechanisms of Cysteine‐Lysine Covalent Linkage—The Role of Reactive Oxygen Species and Competition with Disulfide Bonds**

AbstractRecently, a new naturally occurring covalent linkage was characterised, involving a cysteine and a lysine, bridged through an oxygen atom. The latter was dubbed as the NOS bond, reflecting the individual atoms involved in this uncommon bond which finds little parallel in lab chemistry. It is found to form under oxidising conditions and is reversible upon addition of reducing agents. Further studies have identified the bond in crystal structures across a variety of systems and organisms, potentially playing an important role in regulation, cellular defense and replication. Not only that, double NOS bonds have been identified and even found to be competitive in relation to the formation of disulfide bonds. This raises several questions about how this exotic bond comes to be, what are the intermediates involved in its formation and how it competes with other pathways of sulfide oxidation. With this objective in mind, we revisited our first proposed mechanism for the reaction with model electronic structure calculations, adding information about the reactivity with alternative reactive oxygen species and other potential competing products of oxidation. We present a network with more than 30 reactions which provides one of the most encompassing pictures for cysteine oxidation pathways to date.

Mechanisms of Cysteine-Lysine Covalent Linkage-The Role of Reactive Oxygen Species and Competition with Disulfide Bonds.

Recently, a new naturally occurring covalent linkage was characterised, involving a cysteine and a lysine, bridged through an oxygen atom. The latter was dubbed as the NOS bond, reflecting the individual atoms involved in this uncommon bond which finds little parallel in lab chemistry. It is found to form under oxidising conditions and is reversible upon addition of reducing agents. Further studies have identified the bond in crystal structures across a variety of systems and organisms, potentially playing an important role in regulation, cellular defense and replication. Not only that, double NOS bonds have been identified and even found to be competitive in relation to the formation of disulfide bonds. This raises several questions about how this exotic bond comes to be, what are the intermediates involved in its formation and how it competes with other pathways of sulfide oxidation. With this objective in mind, we revisited our first proposed mechanism for the reaction with model electronic structure calculations, adding information about the reactivity with alternative reactive oxygen species and other potential competing products of oxidation. We present a network with more than 30 reactions which provides one of the most encompassing pictures for cysteine oxidation pathways to date.

Expanded bioinformatic analysis of Oximouse dataset reveals key putative processes involved in brain aging and cognitive decline

The theory that aging is driven by the damage produced by reactive oxygen species (ROS) derived from oxidative metabolism dominated geroscience studies during the second half of the 20th century. However, increasing evidence that ROS also plays a key role in the physiological regulation of numerous processes through the reversible oxidation of cysteine residues in proteins, has challenged this notion. Currently, the scope of redox signaling has reached proteomic dimensions through mass spectrometry techniques. Here, we perform a comprehensive bioinformatics analysis of cysteine oxidation changes during mouse brain aging, using the quantitative data provided in the Oximouse dataset. Interestingly, our unbiased analysis identified hundreds of putative cysteine redox switches covering several pathways previously associated with aging. These include the ubiquitin-proteasome pathway and one-carbon metabolism (folate cycle, methionine cycle, transsulfuration and polyamine pathways). Surprisingly, cysteine oxidation changes are enriched in synaptic proteins in a highly asymmetric distribution: while postsynaptic proteins tend to increase cysteine oxidation with age, the opposite occurs for presynaptic proteins. Additionally, cysteine oxidation changes during aging are associated with proteins involved in the regulation of the mitochondrial transition pore opening and synaptic calcium homeostasis. Our analysis reinforces the concept that brain aging is associated with selective changes in the oxidation state of key proteins, rather than an overall trend toward increased oxidation. Also, we provide a prioritized list of specific cysteine residues with putative impact in aging processes for future experimental validation.

Genome-Wide Identification and Analysis of the Plant Cysteine Oxidase (PCO) Gene Family in Brassica napus and Its Role in Abiotic Stress Response.

Plant Cysteine Oxidase (PCO) is a plant O2-sensing enzyme catalyzing the oxidation of cysteine to Cys-sulfinic acid at the N-termini of target proteins. To better understand the Brassica napus PCO gene family, PCO genes in B. napus and related species were analyzed. In this study, 20, 7 and 8 PCO genes were identified in Brassica napus, Brassica rapa and Brassica oleracea, respectively. According to phylogenetic analysis, the PCOs were divided into five groups: PCO1, PCO2, PCO3, PCO4 and PCO5. Gene organization and motif distribution analysis suggested that the PCO gene family was relatively conserved during evolution. According to the public expression data, PCO genes were expressed in different tissues at different developmental stages. Moreover, qRT-PCR data showed that most of the Bna/Bra/BoPCO5 members were expressed in leaves, roots, flowers and siliques, suggesting an important role in both vegetative and reproductive development. Expression of BnaPCO was induced by various abiotic stress, especially waterlogging stress, which was consistent with the result of cis-element analysis. In this study, the PCO gene family of Brassicaceae was analyzed for the first time, which contributes to a comprehensive understanding of the origin and evolution of PCO genes in Brassicaceae and the function of BnaPCO in abiotic stress responses.

Reaction mechanism for atmospheric pressure plasma treatment of cysteine in solution

Mechanisms for the cold atmospheric plasma (CAP) treatment of cells in solution are needed for more optimum design of plasma devices for wound healing, cancer treatment, and bacterial inactivation. However, the complexity of organic molecules on cell membranes makes understanding mechanisms that result in modifications (i.e. oxidation) of such compounds difficult. As a surrogate to these systems, a reaction mechanism for the oxidation of cysteine in CAP activated water was developed and implemented in a 0-dimensional (plug-flow) global plasma chemistry model with the capability of addressing plasma-liquid interactions. Reaction rate coefficients for organic reactions in water were estimated based on available data in the literature or by analogy to gas-phase reactions. The mechanism was validated by comparison to experimental mass-spectrometry data for COST-jets sustained in He/O2, He/H2O and He/N2/O2 mixtures treating cysteine in water. Results from the model were used to determine the consequences of changing COST-jet operating parameters, such as distance from the substrate and inlet gas composition, on cysteine oxidation product formation. Results indicate that operating parameters can be adjusted to select for desired cysteine oxidation products, including nitrosylated products.

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxICAT, Biotin Switch, and SP3-Rox, these methods typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. Here we establish the local cysteine capture (Cys-LoC) and local cysteine oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole-cell proteomic analysis. Application of the Cys-LOx method to LPS-stimulated immortalized murine bone marrow-derived macrophages (iBMDM), revealed previously unidentified, mitochondrially localized cysteine oxidative modifications upon pro-inflammatory activation, including those associated with oxidative mitochondrial metabolism.

N-Acryloylindole-alkyne (NAIA) enables imaging and profiling new ligandable cysteines and oxidized thiols by chemoproteomics

Cysteine has been exploited as the binding site of covalent drugs. Its high sensitivity to oxidation is also important for regulating cellular processes. To identify new ligandable cysteines which can be hotspots for therapy and to better study cysteine oxidations, we develop cysteine-reactive probes, N-acryloylindole-alkynes (NAIAs), which have superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold. This allows NAIAs to probe functional cysteines more effectively than conventional iodoacetamide-alkyne, and to image oxidized thiols by confocal fluorescence microscopy. In mass spectrometry experiments, NAIAs successfully capture new oxidized cysteines, as well as a new pool of ligandable cysteines and proteins. Competitive activity-based protein profiling experiments further demonstrate the ability of NAIA to discover lead compounds targeting these cysteines and proteins. We show the development of NAIAs with activated acrylamide for advancing proteome-wide profiling and imaging ligandable cysteines and oxidized thiols.

A Single DNA Point Mutation Leads to the Formation of a Cysteine-Tyrosine Crosslink in the Cysteine Dioxygenase from Bacillus subtilis.

Cysteine dioxygenase (CDO) is a non-heme iron-containing enzyme that catalyzes the oxidation of cysteine (Cys) to cysteine sulfinic acid (CSA). Crystal structures of eukaryotic CDOs revealed the presence of an unusual crosslink between the sulfur of a cysteine residue (C93 in Mus musculus CDO, MmCDO) and a carbon atom adjacent to the phenyl group of a tyrosine residue (Y157). Formation of this crosslink occurs over time as a byproduct of catalysis and increases the catalytic efficiency of CDO by at least 10-fold. Interestingly, in bacterial CDOs, the residue corresponding to C93 is replaced by a highly conserved glycine (G82 in Bacillus subtilis CDO, BsCDO), which precludes the formation of a C-Y crosslink in these enzymes; yet bacterial CDOs achieve turnover rates paralleling those of fully crosslinked eukaryotic CDOs. In the present study, we prepared the G82C variant of BsCDO to determine if a single DNA point mutation could lead to C-Y crosslink formation in this enzyme. We used gel electrophoresis, peptide mass spectrometry, electron paramagnetic resonance spectroscopy, and kinetic assays to characterize this variant alongside the natively crosslinked wild-type (WT) MmCDO and the natively non-crosslinked WT BsCDO. Collectively, our results provide compelling evidence that the G82C BsCDO variant is indeed capable of C-Y crosslink formation. Our kinetic studies indicate that G82C BsCDO has a reduced catalytic efficiency compared to WT BsCDO and that activity increases as the ratio of crosslinked to non-crosslinked enzyme increases. Finally, by carrying out a bioinformatic analysis of the CDO family, we were able to identify a large number of putatively crosslinked bacterial CDOs, the majority of which are from Gram-negative pathogenic bacteria.

Nitric oxide delivery and heme-assisted S-nitrosation by the bedbug nitrophorin

Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect's salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (Kd = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.

In silico exploration of antioxidants as oxidation protectant for PITRM1 peptidase activity, an Alzheimer disease target.

To find efficient antioxidants to protect oxidation prone cysteine residues of the peptidase PITRM1 using molecular docking and simulation techniques. A total of 50 antioxidants were docked with PITRM1 at the oxidation prone region Cys89 and Cys96 using Autodock Vina software. The lowest socred compounds were predicted for its Blood brain barrier permeability using LightBBB. Molecular dynamic simulations of the PITRM1 and Ascorbic acid/Silymarin complex were performed using the GROMACS 2020.1 package and the free energy calculations were performed using gmx_MMPBSA. The RMSD, RMSF, Rg, Minimum distance and Hydrogen bonds were also evaluated. Silymarin, Ascorbic acid, Naringenin, Gallic acid, Chlorogenic acid, Rosmarinic acid, (-)-Epicatechin, Genistein showed a docking score of above -5.3 kcal/mol. Silymarin and Ascorbic acid were predicted to cross the Blood Brain Barrier. Molecular dynamic simulation and mmPBSA analysis revealed that Silymarin showed a positive free energy implying no affinity to PITRM1 and ascorbic acid has low ΔG with -13.13 kJ/mol. The stability of the ascorbic acid complex was high (RMSD: 0.160 ± 0.018 nm, Minimum Distance: 0.163 ± 0.001 nm and four hydrogen bonds) and fluctuation induced due to ascorbic acid was low. Ascorbic acid was found to effectively interact with the cysteine oxidation prone region and can have a potential role in reducing the oxidised cysteine in PITRM1 to modulate its peptidase activity.

Oxidization of optic atrophy 1 cysteines occurs during heart ischemia-reperfusion and amplifies cell death by oxidative stress

During cardiac ischemia-reperfusion, excess reactive oxygen species can damage mitochondrial, cellular and organ function. Here we show that cysteine oxidation of the mitochondrial protein Opa1 contributes to mitochondrial damage and cell death caused by oxidative stress. Oxy-proteomics of ischemic-reperfused hearts reveal oxidation of the C-terminal C786 of Opa1 and treatment of perfused mouse hearts, adult cardiomyocytes, and fibroblasts with H2O2 leads to the formation of a reduction-sensitive ∼180 KDa Opa1 complex, distinct from the ∼270 KDa one antagonizing cristae remodeling. This Opa1 oxidation process is curtailed by mutation of C786 and of the other 3 Cys residues of its C-terminal domain (Opa1TetraCys). When reintroduced in Opa1−/− cells, Opa1TetraCys is not efficiently processed into short Opa1TetraCys and hence fails to fuse mitochondria. Unexpectedly, Opa1TetraCys restores mitochondrial ultrastructure in Opa1−/− cells and protects them from H2O2-induced mitochondrial depolarization, cristae remodeling, cytochrome c release and cell death. Thus, preventing the Opa1 oxidation occurring during cardiac ischemia-reperfusion reduces mitochondrial damage and cell death induced by oxidative stress independent of mitochondrial fusion.