Redox Proteomics Research Articles

Abstract 4136813: Redox Serum Proteomics Of Left Ventricular Assist Device Unloaded Human Heart

Heart failure (HF) is a global epidemic claiming millions of lives worldwide. Unloading the heart of end-stage HF patients via left ventricular assist devices (LVADs) is used not only as a bridge to transplantation but also as a bridge to recovery in certain selected patients. To date, assessment of patients’ recovery from HF is based on clinical and symptomatic evaluation. The use of non-invasive biomarkers that monitor the remodeling/reverse remodeling associated with HF progression/recovery is still lacking. In this study we applied mass spectrometry-based quantitative redox proteomics to identify serum biomarkers to monitor both the progression of HF and its recovery after LVAD treatment. A fingerprint of reversed behavior at the protein and at the oxidized Cys peptide level has been discovered when comparing HF and LVAD patients. HDL proteins related to lipid metabolism were decreased in HF samples and increased in LVAD patients. In addition, a number of inflammatory proteins showed a decrease in LVAD samples and an increase in HF samples. In particular APOA4, C7, F2 or SAA2 discriminated between HF and LVAD patients. A pool of peptides containing specific oxidized Cys residues showed an inverse relationship in HF as opposed to LVAD samples. Oxidized Cys-containing peptides derived from Albumin, IGK and IGG1 discriminated HF from LVAD samples. Discriminatory sensitivity and specificity were enhanced by using a combination of the proteins APOA4, C7, F2 and the above-mentioned peptide derived from Albumin. Responders and non-responders could also be separated by proteins such as APOA4, and Cys-oxidized peptides, such as the ones derived from Albumin or IGK mentioned above. This is the first study to apply redox proteomics to explore the protein profile of the LVAD unloaded human heart. These data suggest that HDL plays an important role in LVAD recovery and that a specific group of peptides is associated with the recovery process; the mechanisms involved need further study. Our data could have important implications to the management of patients in the LVAD recovery program and could serve to identify new therapeutic targets.

Redox proteomic analysis of H2O2 -treated Jurkat cells and effects of bicarbonate and knockout of peroxiredoxins 1 and 2

Oxidation of thiol proteins and redox signaling occurs in cells exposed to H2O2 but mechanisms are unclear. We used redox proteomics to seek evidence of oxidation of specific proteins either by a mechanism involving reaction of H2O2 with CO2/bicarbonate to give the more reactive peroxymonocarbonate, or via a relay involving peroxiredoxins (Prdxs). Changes in oxidation state of specific Cys-SH residues on treating Jurkat T lymphoma cells with H2O2 were measured by isotopically labeling reduced thiols and analysis by mass spectrometry. The effects of bicarbonate and of knocking out either Prdx1 or Prdx2 were examined. Approximately 14,000 Cys-peptides were detected, of which ∼ 1% underwent 2-10 fold loss in thiol content with H2O2. Those showing the most oxidation were not affected by the presence of bicarbonate or knockout of either Prdx. Consistent with previous evidence that bicarbonate potentiates inactivation of glyceraldehyde-3-phosphate dehydrogenase, the GAPDH active site Cys residues were significantly more sensitive to H2O2 when bicarbonate was present. Several other proteins were identified as promising candidates for further investigation. Although we identified some potential protein candidates for Prdx- dependent oxidation, most of the significant differences between KO and WT cells were seen in proteins for which H2O2 unexpectedly increased their CysSH content over untreated cells. We conclude that facilitation of protein oxidation by bicarbonate or Prdx-mediated relays is restricted to a small number of proteins and is insufficient to explain the majority of the oxidation of cell thiols that occurs in response to H2O2.

Chemoproteogenomic stratification of the missense variant cysteinome

Cancer genomes are rife with genetic variants; one key outcome of this variation is widespread gain-of-cysteine mutations. These acquired cysteines can be both driver mutations and sites targeted by precision therapies. However, despite their ubiquity, nearly all acquired cysteines remain unidentified via chemoproteomics; identification is a critical step to enable functional analysis, including assessment of potential druggability and susceptibility to oxidation. Here, we pair cysteine chemoproteomics—a technique that enables proteome-wide pinpointing of functional, redox sensitive, and potentially druggable residues—with genomics to reveal the hidden landscape of cysteine genetic variation. Our chemoproteogenomics platform integrates chemoproteomic, whole exome, and RNA-seq data, with a customized two-stage false discovery rate (FDR) error controlled proteomic search, which is further enhanced with a user-friendly FragPipe interface. Chemoproteogenomics analysis reveals that cysteine acquisition is a ubiquitous feature of both healthy and cancer genomes that is further elevated in the context of decreased DNA repair. Reference cysteines proximal to missense variants are also found to be pervasive, supporting heretofore untapped opportunities for variant-specific chemical probe development campaigns. As chemoproteogenomics is further distinguished by sample-matched combinatorial variant databases and is compatible with redox proteomics and small molecule screening, we expect widespread utility in guiding proteoform-specific biology and therapeutic discovery.

Accumulation, biochemical responses and changes in the redox proteome promoted by Ag and Cd in the burrowing bivalve Scrobicularia plana

Silver (Ag) and cadmium (Cd) are non-essential metals that, as a result of natural processes and human activities, reach the aquatic environment where they interact with biota inducing potential toxic effects. To determine the biological effects of these metals on the endobenthic bivalve Scrobicularia plana, specimens were exposed to Ag and Cd at two concentrations, 5 and 50 μg∙L-1, for 7 days in a controlled microcosm system. The levels of the metals were measured in the seawater, sediments and clam tissues. The possible toxic biological effects of Ag and Cd were studied using a battery of biochemical biomarkers that are responsive to oxidative stress: superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST) activities, and metallothioneins (MTs) and lipid peroxidation (LPO) levels. Since both metals have been linked to oxidative stress, redox modifications to proteins were studied by differential isotopic labelling of the oxidised and reduced forms of cysteines (Cys). An accumulation of metals was observed in the digestive gland and gills following exposure, together with the activation of enzyme activities (SOD for the Cd exposure; SOD, CAT, GST, and GR for the Ag exposure). The MT and LPO levels (after individual exposure to Ag and Cd) increased, which suggests the existence of antioxidant and detoxification processes to mitigate the toxic oxidative effects of both metals. The redox proteomic analysis identified 771 Cys-containing peptides (out of 514 proteins), of which 195 and 226 changed after exposure to Ag and Cd, respectively. Bioinformatics analysis showed that exposure to metal affects relevant functional pathways and biological processes in S. plana, such as: “cellular respiration” (Ag), “metabolism of amino acids” and “synthesis and degradation of proteins” (Ag and Cd), “carbohydrate metabolism” and “oxidative stress” (Cd). The proteomic approach implemented here is a powerful complement to conventional biochemical biomarkers, since it evaluates changes at the protein level in a high-throughput unbiased manner, thus providing a general appraisal of the biological responses altered by exposure to the contaminants.

Exploring oxysterols and protein carbonylation in cervicovaginal secretions as biomarkers for cervical cancer development

Cervical cancer, a major global health issue and the fourth most common cancer among women, is strongly linked to Human Papillomavirus (HPV) infection. Emerging evidence indicates that oxidative stress plays a critical role in the carcinogenesis of cervical tissue. This study investigates the relationship between oxidative stress markers—specifically oxysterols, lipid oxidation, and protein carbonylation—and the progression of cervical neoplasia.Oxysterols, which are elevated in various inflammatory diseases and cancers, were measured in cervicovaginal fluid samples using LC-MS/MS. The targeted oxysterols included 27-hydroxycholesterol (27-OHC), 7β-hydroxycholesterol (7β-OHC), 7-ketocholesterol (7-KC), and 7α,27-dihydroxycholesterol (7α,27-diOHC). Among these, 7α,27-dihydroxycholesterol was significantly increased in correlation with the severity of neoplastic stages. In parallel, protein carbonylation, an indicator of cellular oxidative stress, was assessed. Results revealed higher levels of protein carbonylation in neoplastic samples compared to non-neoplastic controls. These modifications were further analysed through redox proteomics to identify the specific proteins affected.The study demonstrates that elevated lipid oxidation and protein carbonylation in cervicovaginal secretions are linked to the development and progression of cervical cancer. Identifying these biomarkers may improve screening strategies, enabling the identification of individuals at increased risk for cervical neoplasia and guiding timely interventions.

Multifaceted Proteome Analysis at Solubility, Redox, and Expression Dimensions for Target Identification.

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, the first high-throughput redox proteomics approach integrated with expression analysis (REX) is devised and combined with the Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, HCT116 cells treated with auranofin are analyzed, showing great improvement compared with previous studies. PISA-REX is then applied to study proteome remodeling upon stimulation of human monocytes by interferon α (IFN-α). Applying this tool to study the proteome changes in plasmacytoid dendritic cells (pDCs) isolated from wild-type versus Ncf1-mutant mice treated with interferon α, shows that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility, and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

Oxidative Cysteine Post Translational Modifications Drive the Redox Code Underlying Neurodegeneration and Amyotrophic Lateral Sclerosis.

Redox dysregulation, an imbalance between oxidants and antioxidants, is crucial in the pathogenesis of various neurodegenerative diseases. Within this context, the "redoxome" encompasses the network of redox molecules collaborating to maintain cellular redox balance and signaling. Among these, cysteine-sensitive proteins are fundamental for this homeostasis. Due to their reactive thiol groups, cysteine (Cys) residues are particularly susceptible to oxidative post-translational modifications (PTMs) induced by free radicals (reactive oxygen, nitrogen, and sulfur species) which profoundly affect protein functions. Cys-PTMs, forming what is referred to as "cysteinet" in the redox proteome, are essential for redox signaling in both physiological and pathological conditions, including neurodegeneration. Such modifications significantly influence protein misfolding and aggregation, key hallmarks of neurodegenerative diseases such as Alzheimer's, Parkinson's, and notably, amyotrophic lateral sclerosis (ALS). This review aims to explore the complex landscape of cysteine PTMs in the cellular redox environment, elucidating their impact on neurodegeneration at protein level. By investigating specific cysteine-sensitive proteins and the regulatory networks involved, particular emphasis is placed on the link between redox dysregulation and ALS, highlighting this pathology as a prime example of a neurodegenerative disease wherein such redox dysregulation is a distinct hallmark.

Unveiling novel targets in melanoma under melanogenesis stimulation and photodynamic therapy by redox proteomics.

Melanogenesis-stimulated B16-F10 cells enter in a quiescent state, present inhibited mitochondrial respiration and increased reactive oxygen species levels. These alterations suggest that these cells may be under redox signaling, allowing tumor survival. The aim of this study was to evaluate redox-modified proteins in B16-F10 cells after melanogenesis stimulation and rose bengal-photodynamic therapy (RB-PDT). A redox proteomics label-free approach based on the biotin switch assay technique with biotin-HPDP and N-ethylmaleimide was used to assess the thiol-oxidized protein profile. Aconitase was oxidized at Cys-448 and Cys-451, citrate synthase was oxidized at Cys-202 and aspartate aminotransferase (Got2) was oxidized at Cys-272 and Cys-274, exclusively after melanogenesis stimulation. After RB-PDT, only guanine nucleotide-binding protein subunit beta-2-like 1 (Gnb2l1) was oxidized (Cys-168). In contrast, melanogenesis stimulation followed by RB-PDT led to the oxidation of different cysteines in Gnb2l1 (Cys-153 and Cys-249). Besides that, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) presented oxidation at Cys-245, peptidyl-prolyl cis-trans isomerase A (Ppia) was oxidized at Cys-161 and 5,6-dihydroxyindole-2-carboxylic acid oxidase (Tyrp1) was oxidized at Cys-65, Cys-30, and Cys-336 after melanogenesis stimulation followed by RB-PDT. The redox alterations observed in murine melanoma cells and identification of possible target proteins are of great importance to further understand tumor resistance mechanisms.

Unlocking the molecular modifications of plasma-activated water-induced oxidation through redox proteomics: In the case of duck myofibrillar protein (Anas platyrhynchos)

Plasma-activated water (PAW) contains multiple active species that alter the structure of myofibrillar protein (MP) to enhance their gel properties. This work investigated the impact of PAW on the oxidation of cysteine in MP by label-free quantitative proteomics. PAW treatment caused the oxidation of 8241 cysteine sites on 2815 proteins, and structural proteins such as nebulin, myosin XVIIIB, myosin XVIIIA, and myosin heavy chain were susceptible to oxidation by PAW. Bioinformatics analysis, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, subcellular localization, and STRING analysis, indicated that these proteins with differential oxidation sites were mainly derived from the cytoplasm and membrane, and were involved in multiple GO terms and KEGG pathways. This is one of the first reports of the redox proteomic changes induced by PAW treatment, and the results are useful for understanding the possible mechanism of PAW-induced oxidation of MP.

Redox proteomics in melanoma cells: An optimized protocol

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.

Redox proteomics of PANC-1 cells reveals the significance of HIF-1 signaling protein oxidation in pancreatic ductal adenocarcinoma pathogenesis

BackgroundProtein cysteine oxidation is substantially involved in various biological and pathogenic processes, but its implications in pancreatic cancer development remains poorly understood.Methods and resultsIn this study, we performed a global characterization of protein oxidation targets in PDAC cells through iodoTMT-based quantitative proteomics, which identified over 4300 oxidized cysteine sites in more than 2100 proteins in HPDE6c7 and PANC-1 cells. Among them, 1715 cysteine residues were shown to be differentially oxidized between HPDE6c7 and PANC-1 cells. Also, charged amino acids including aspartate, glutamate and lysine were significantly overrepresented in flanking sequences of oxidized cysteines. Differentially oxidized proteins in PANC-1 cells were enriched in multiple cancer-related biological processes and signaling pathways. Specifically, the HIF-1 signaling proteins exhibited significant oxidation alterations in PANC-1 cells, and the reduced PHD2 oxidation in human PDAC tissues was correlated with lower survival time in pancreatic cancer patients.ConclusionThese investigations provided new insights into protein oxidation-regulated signaling and biological processes during PDAC pathogenesis, which might be further explored for pancreatic cancer diagnosis and treatment.

Methionine oxidation of carbohydrate-active enzymes during white-rot wood decay.

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown.

Signatures of cysteine oxidation on muscle structural and contractile proteins are associated with physical performance and muscle function in older adults: Study of Muscle, Mobility and Aging (SOMMA).

Oxidative stress is considered a contributor to declining muscle function and mobility during aging; however, the underlying molecular mechanisms remain poorly described. We hypothesized that greater levels of cysteine (Cys) oxidation on muscle proteins are associated with decreased measures of mobility. Herein, we applied a novel redox proteomics approach to measure reversible protein Cys oxidation in vastus lateralis muscle biopsies collected from 56 subjects in the Study of Muscle, Mobility and Aging (SOMMA), a community-based cohort study of individuals aged 70 years and older. We tested whether levels of Cys oxidation on key muscle proteins involved in muscle structure and contraction were associated with muscle function (leg power and strength), walking speed, and fitness (VO2 peak on cardiopulmonary exercise testing) using linear regression models adjusted for age, sex, and body weight. Higher oxidation levels of select nebulin Cys sites were associated with lower VO2 peak, while greater oxidation of myomesin-1, myomesin-2, and nebulin Cys sites was associated with slower walking speed. Higher oxidation of Cys sites in key proteins such as myomesin-2, alpha-actinin-2, and skeletal muscle alpha-actin were associated with lower leg power and strength. We also observed an unexpected correlation (R = 0.48) between a higher oxidation level of eight Cys sites in alpha-actinin-3 and stronger leg power. Despite this observation, the results generally support the hypothesis that Cys oxidation of muscle proteins impairs muscle power and strength, walking speed, and cardiopulmonary fitness with aging.

Using Redox Proteomics to Gain New Insights into Neurodegenerative Disease and Protein Modification.

Antioxidant defenses in biological systems ensure redox homeostasis, regulating baseline levels of reactive oxygen and nitrogen species (ROS and RNS). Oxidative stress (OS), characterized by a lack of antioxidant defenses or an elevation in ROS and RNS, may cause a modification of biomolecules, ROS being primarily absorbed by proteins. As a result of both genome and environment interactions, proteomics provides complete information about a cell's proteome, which changes continuously. Besides measuring protein expression levels, proteomics can also be used to identify protein modifications, localizations, the effects of added agents, and the interactions between proteins. Several oxidative processes are frequently used to modify proteins post-translationally, including carbonylation, oxidation of amino acid side chains, glycation, or lipid peroxidation, which produces highly reactive alkenals. Reactive alkenals, such as 4-hydroxy-2-nonenal, are added to cysteine (Cys), lysine (Lys), or histidine (His) residues by a Michael addition, and tyrosine (Tyr) residues are nitrated and Cys residues are nitrosylated by a Michael addition. Oxidative and nitrosative stress have been implicated in many neurodegenerative diseases as a result of oxidative damage to the brain, which may be especially vulnerable due to the large consumption of dioxygen. Therefore, the current methods applied for the detection, identification, and quantification in redox proteomics are of great interest. This review describes the main protein modifications classified as chemical reactions. Finally, we discuss the importance of redox proteomics to health and describe the analytical methods used in redox proteomics.

The impact of light and thioredoxins on the plant thiol-disulfide proteome.

Thiol-based redox regulation is a crucial posttranslational mechanism to acclimate plants to changing light availability. Here, we conducted a biotin switch-based redox proteomics study in Arabidopsis (Arabidopsis thaliana) to systematically investigate dynamics of thiol-redox networks in response to temporal changes in light availability and across genotypes lacking parts of the thioredoxin (Trx) or NADPH-Trx-reductase C (NTRC) systems in the chloroplast. Time-resolved dynamics revealed light led to marked decreases in the oxidation states of many chloroplast proteins with photosynthetic functions during the first 10 min, followed by their partial reoxidation after 2 to 6 h into the photoperiod. This involved f, m, and x-type Trx proteins showing similar light-induced reduction-oxidation dynamics, while NTRC, 2-Cys peroxiredoxins, and Trx y2 showed an opposing pattern, being more oxidized in light than dark. In Arabidopsis trxf1f2, trxm1m2, or ntrc mutants, most proteins showed increased oxidation states in the light compared to wild type, suggesting their light-dependent dynamics were related to NTRC/Trx networks. While NTRC deficiency had a strong influence in all light conditions, deficiencies in f- or m-type Trxs showed differential impacts on the thiol-redox proteome depending on the light environment, being higher in constant or fluctuating light, respectively. The results indicate plant redox proteomes are subject to dynamic changes in reductive and oxidative pathways to cooperatively fine-tune photosynthetic and metabolic processes in the light. The importance of the individual elements of the NTRC/Trx networks mediating these responses depend on the extent of light variability, with NTRC playing a crucial role to balance protein-redox states in rapidly fluctuating light.

Oxidative damage in neurodegeneration: roles in the pathogenesis and progression of Alzheimer disease.

Alzheimer disease (AD) is associated with multiple etiologies and pathological mechanisms, among which oxidative stress (OS) appears as a major determinant. Intriguingly, OS arises in various pathways regulating brain functions, and it seems to link different hypotheses and mechanisms of AD neuropathology with high fidelity. The brain is particularly vulnerable to oxidative damage, mainly because of its unique lipid composition, resulting in an amplified cascade of redox reactions that target several cellular components/functions ultimately leading to neurodegeneration. The present review highlights the "OS hypothesis of AD," including amyloid beta-peptide-associated mechanisms, the role of lipid and protein oxidation unraveled by redox proteomics, and the antioxidant strategies that have been investigated to modulate the progression of AD. Collected studies from our groups and others have contributed to unraveling the close relationships between perturbation of redox homeostasis in the brain and AD neuropathology by elucidating redox-regulated events potentially involved in both the pathogenesis and progression of AD. However, the complexity of AD pathological mechanisms requires an in-depth understanding of several major intracellular pathways affecting redox homeostasis and relevant for brain functions. This understanding is crucial to developing pharmacological strategies targeting OS-mediated toxicity that may potentially contribute to slow AD progression as well as improve the quality of life of persons with this severe dementing disorder.

Thiol Redox Proteomics for Identifying Redox-Sensitive Cysteine Residues Within the Protein of Interest During Stress.

Redox modulation is a common posttranslational modification to regulate protein activity. The targets of oxidizing agents are cysteine residues(Cys), which have to be exposed at the surface of the proteins and are characterized by an environment that favors redox modulation. This includes their protonation state and the neighboring amino acids. The Cys redox state can be assessed experimentally by redox titrations to determine the midpoint redox potential in the protein. Exposed cysteine residues and putative intramolecular disulfide bonds can be predicted by alignments with structural data using dedicated software tools and information on conserved cysteine residues. Labeling with light and heavy reagents, such as N-ethylmaleimide (NEM), followed by mass spectrometric analysis, allows for the experimental determination of redox-responsive cysteine residues. This type of thiol redox proteomics is a powerful approach to assessing the redox state of the cell, e.g., in dependence on environmental conditions and, in particular, under abiotic stress.

The redox code of plants.

Central metabolism is organised through high-flux, Nicotinamide Adenine Dinucleotide(NAD+/NADH) and NADP+/NADPH systems operating at near equilibrium. As oxygen is indispensable for aerobic organisms, these systems are also linked to the levels of reactive oxygen species, such as H2O2, and through H2O2 to the regulation of macromolecular structures and activities, via kinetically controlled sulphur switches in the redox proteome. Dynamic changes in H2O2 production, scavengingand transport, associated with development, growthand responses to the environment are, therefore, linked to the redox state of the cell and regulate cellular function. These basic principles form the 'redox code' of cells and were first defined by D. P. Jones and H. Sies in 2015. Here, we apply these principles to plants in which recent studies have shown that they can also explain cell-to-cell and even plant-to-plant signalling processes. The redox code is, therefore, an integral part of biological systems and can be used to explain multiple processes in plants at the subcellular, cellular, tissue, whole organismand perhaps even community and ecosystem levels. As the environmental conditions on our planet are worsening due to global warming, climate changeand increased pollution levels, new studies are needed applying the redox code of plants to these changes.

Harnessing redox proteomics to study metabolic regulation and stress response in lignin-fed Rhodococci

BackgroundRhodococci are studied for their bacterial ligninolytic capabilities and proclivity to accumulate lipids. Lignin utilization is a resource intensive process requiring a variety of redox active enzymes and cofactors for degradation as well as defense against the resulting toxic byproducts and oxidative conditions. Studying enzyme expression and regulation between carbon sources will help decode the metabolic rewiring that stymies lignin to lipid conversion in these bacteria. Herein, a redox proteomics approach was applied to investigate a fundamental driver of carbon catabolism and lipid anabolism: redox balance.ResultsA consortium of Rhodococcus strains was employed in this study given its higher capacity for lignin degradation compared to monocultures. This consortium was grown on glucose vs. lignin under nitrogen limitation to study the importance of redox balance as it relates to nutrient availability. A modified bottom–up proteomics workflow was harnessed to acquire a general relationship between protein abundance and protein redox states. Global proteomics results affirm differential expression of enzymes involved in sugar metabolism vs. those involved in lignin degradation and aromatics metabolism. As reported previously, several enzymes in the lipid biosynthetic pathways were downregulated, whereas many involved in β-oxidation were upregulated. Interestingly, proteins involved in oxidative stress response were also upregulated perhaps in response to lignin degradation and aromatics catabolism, which require oxygen and reactive oxygen species and generate toxic byproducts. Enzymes displaying little-to-no change in abundance but differences in redox state were observed in various pathways for carbon utilization (e.g., β‑ketoadipate pathway), lipid metabolism, as well as nitrogen metabolism (e.g., purine scavenging/synthesis), suggesting potential mechanisms of redox-dependent regulation of metabolism.ConclusionsEfficient lipid production requires a steady carbon and energy flux while balancing fundamental requirements for enzyme production and cell maintenance. For lignin, we theorize that this balance is difficult to establish due to resource expenditure for enzyme production and stress response. This is supported by significant changes to protein abundances and protein cysteine oxidation in various metabolic pathways and redox processes.

PTM-Psi: A python package to facilitate the computational investigation of post-translational modification on protein structures and their impacts on dynamics and functions.

Post-translational modification (PTM) of a protein occurs after it has been synthesized from its genetic template, and involves chemical modifications of the protein's specific amino acid residues. Despite of the central role played by PTM in regulating molecular interactions, particularly those driven by reversible redox reactions, it remains challenging to interpret PTMs in terms of protein dynamics and function because there are numerous combinatorially enormous means for modifying amino acids in response to changes in the protein environment. In this study, we provide a workflow that allows users to interpret how perturbations caused by PTMs affect a protein's properties, dynamics, and interactions with its binding partners based on inferred or experimentally determined protein structure. This Python-based workflow, called PTM-Psi, integrates several established open-source software packages, thereby enabling the user to infer protein structure from sequence, develop force fields for non-standard amino acids using quantum mechanics, calculate free energy perturbations through molecular dynamics simulations, and score the bound complexes via docking algorithms. Using the S-nitrosylation of several cysteines on the GAP2 protein as an example, we demonstrated the utility of PTM-Psi for interpreting sequence-structure-function relationships derived from thiol redox proteomics data. We demonstrate that the S-nitrosylated cysteine that is exposed to the solvent indirectly affects the catalytic reaction of another buried cysteine over a distance in GAP2 protein through the movement of the two ligands. Our workflow tracks the PTMs on residues that are responsive to changes in the redox environment and lays the foundation for the automation of molecular and systems biology modeling.