Regulation of the redox metabolome and thiol proteome by hydrogen sulfide.

Overproduction of reactive oxygen species and compromised antioxidant defenses perturb intracellular redox homeostasis and is associated with a myriad of human diseases as well as with the natural process of aging. Hydrogen sulfide (H2S), which is biosynthesized by organisms ranging from bacteria to man, influences a broad range of physiological functions. A highly touted molecular mechanism by which H2S exerts its cellular effects is via post-translational modification of the thiol redox proteome, converting cysteine thiols to persulfides, in a process referred to as protein persulfidation. The physiological relevance of this modification in the context of specific signal transmission pathways remains to be rigorously established, while a general protective role for protein persulfidation against hyper-oxidation of the cysteine proteome is better supported. A second mechanism by which H2S modulates redox homeostasis is via remodeling the redox metabolome, targeting the electron transfer chain and perturbing the major redox nodes i.e. CoQ/CoQH2, NAD+/NADH and FAD/FADH2. The metabolic changes that result from H2S-induced redox changes fan out from the mitochondrion to other compartments. In this review, we discuss recent developments in elucidating the roles of H2S and its oxidation products on redox homeostasis and its role in protecting the thiol proteome.

Thiol redox proteomics: Characterization of thiol-based post-translational modifications.

Redox post-translational modifications on cysteine thiols (redox PTMs) have profound effects on protein structure and function, thus enabling regulation of various biological processes. Redox proteomics approaches aim to characterize the landscape of redox PTMs at the systems level. These approaches facilitate studies of condition-specific, dynamic processes implicating redox PTMs and have furthered our understanding of redox signaling and regulation. Mass spectrometry (MS) is a powerful tool for such analyses which has been demonstrated by significant advances in redox proteomics during the last decade. A group of well-established approaches involves the initial blocking of free thiols followed by selective reduction of oxidized PTMs and subsequent enrichment for downstream detection. Alternatively, novel chemoselective probe-based approaches have been developed for various redox PTMs. Direct detection of redox PTMs without any enrichment has also been demonstrated given the sensitivity of contemporary MS instruments. This review discusses the general principles behind different analytical strategies and covers recent advances in redox proteomics. Several applications of redox proteomics are also highlighted to illustrate how large-scale redox proteomics data can lead to novel biological insights.

Evaluation of linear models and missing value imputation for the analysis of peptide-centric proteomics.

BACKGROUND: Several methods to handle data generated from bottom-up proteomics via liquid chromatography-mass spectrometry, particularly for peptide-centric quantification dealing with post-translational modification (PTM) analysis like reversible cysteine oxidation are evaluated. The paper proposes a pipeline based on the R programming language to analyze PTMs from peptide-centric label-free quantitative proteomics data. RESULTS: Our methodology includes variance stabilization, normalization, and missing data imputation to account for the large dynamic range of PTM measurements. It also corrects biases from an enrichment protocol and reduces the random and systematic errors associated with label-free quantification. The performance of the methodology is tested by performing proteome-wide differential PTM quantitation using linear models analysis (limma). We objectively compare two imputation methods along with significance testing when using multiple-imputation for missing data. CONCLUSION: Identifying PTMs in large-scale datasets is a problem with distinct characteristics that require new methods for handling missing data imputation and differential proteome analysis. Linear models in combination with multiple-imputation could significantly outperform a t-test-based decision method.

Global Thiol Proteome Analysis Provides Novel Insights into the Macrophage Inflammatory Response and Its Regulation by the Thioredoxin System.

Aims: Oxidative modifications of cysteine (Cys) thiols regulate various physiological processes, including inflammatory responses. The thioredoxin (Trx) system plays a key role in thiol redox control. The aim of this study was to characterize the dynamic cysteine proteome of human macrophages upon activation by the prototypical proinflammatory agent, bacterial lipopolysaccharide (LPS), and/or perturbation of the Trx system. Results: In this study, we profiled the cellular and redox proteome of human THP-1-derived macrophages during the early phase of LPS activation and/or inhibition of Trx system activity by auranofin (AF) by employing a peptide-centric, resin-assisted capture, redox proteomic workflow. Among 4200 identified cysteines, oxidation of nearly 10% was selectively affected by LPS or AF treatments. Notably, the proteomic analysis uncovered a subset of ∼100 thiols, mapped to proteins involved in diverse processes, whose oxidation is antagonistically regulated by LPS and Trx. Compared with the redox proteome, the cellular proteome was largely unchanged, highlighting the importance of redox modification as a mechanism that allows for rapid modulation of macrophage activities in response to a proinflammatory or pro-oxidant insult. Structural-functional analyses provided mechanistic insights into redox regulation of selected proteins, including the glutathione-synthesizing enzyme, glutamate-cysteine ligase, and the autophagy adaptor, SQSTM1/p62, suggesting mechanisms by which macrophages adapt and fine-tune their responses according to a changing inflammatory and redox environment. Innovation: This study provides a rich resource for further characterization of redox mechanisms that regulate macrophage inflammatory activities. Conclusion: The dynamic thiol redox proteome allows macrophages to efficiently respond and adapt to redox and inflammatory challenges. Antioxid. Redox Signal. 38, 388-402.

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.

SP3-FAIMS-Enabled High-Throughput Quantitative Profiling of the Cysteinome.

Cysteine-directed chemoproteomic profiling methods yield high-throughput inventories of redox-sensitive and ligandable cysteine residues and therefore are enabling techniques for functional biology and drug discovery. However, the cumbersome nature of many sample preparation workflows, the requirements for large amounts of input material, and the modest yields of labeled peptides are limitations that hinder most chemoproteomics studies. Here, we report an optimized chemoproteomic sample-preparation workflow that combines enhanced peptide labeling with single-pot, solid-phase-enhanced sample preparation (SP3) to improve the recovery of biotinylated peptides, even from small samples. We further tailor our SP3 method to specifically probe the redox proteome, which showcases the utility of the SP3 platform in multistep sample-preparation workflows. By implementing a customized workflow in the FragPipe computational pipeline, we achieve accurate MS1-based quantification, including for peptides containing multiple cysteine residues. Collectively these innovations enable enhanced high-throughput quantitative analysis of the cysteinome. This article includes detailed protocols for cysteine labeling with isotopically labeled iodoacetamide alkyne probes, biotinylation with CuAAC, sample cleanup with SP3, enrichment of cysteines with NeutrAvidin agarose beads, LC-FAIMS-MS/MS analysis, and FragPipe-IonQuant analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling of cysteines in human proteome and SP3-based sample cleanup Alternate Protocol 1: Labeling of cysteines in human proteome, SP3-based sample cleanup, and enrichment of cysteines for isoTOP-ABPP analysis Alternate Protocol 2: Labeling of cysteines in human proteome and SP3-based sample cleanup for redox proteome analysis Basic Protocol 2: Peptide-level cysteine enrichment Basic Protocol 3: LC-FAIMS-MS/MS analysis Basic Protocol 4: FragPipe data analysis.

The Redox Proteome of Thiol Proteins in the Rice Blast Fungus Magnaporthe oryzae.

Redox modification, a post-translational modification, has been demonstrated to be significant for many physiological pathways and biological processes in both eukaryotes and prokaryotes. However, little is known about the global profile of protein redox modification in fungi. To explore the roles of redox modification in the plant pathogenic fungi, a global thiol proteome survey was performed in the model fungal pathogen Magnaporthe oryzae. A total of 3713 redox modification sites from 1899 proteins were identified through a mix sample containing mycelia with or without oxidative stress, conidia, appressoria, and invasive hyphae of M. oryzae. The identified thiol-modified proteins were performed with protein domain, subcellular localization, functional classification, metabolic pathways, and protein-protein interaction network analyses, indicating that redox modification is associated with a wide range of biological and cellular functions. These results suggested that redox modification plays important roles in fungal growth, conidium formation, appressorium formation, as well as invasive growth. Interestingly, a large number of pathogenesis-related proteins were redox modification targets, suggesting the significant roles of redox modification in pathogenicity of M. oryzae. This work provides a global insight into the redox proteome of the pathogenic fungi, which built a groundwork and valuable resource for future studies of redox modification in fungi.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation.

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women.

Knockout of PRDX6 induces mitochondrial dysfunction and cell cycle arrest at G2/M in HepG2 hepatocarcinoma cells.

Peroxiredoxin 6 (PRDX6) has been associated with tumor progression and cancer metastasis. Its acting on phospholipid hydroperoxides and its phospholipase-A2 activity are unique among the peroxiredoxin family and add complexity to its action mechanisms. As a first step towards the study of PRDX6 involvement in cancer, we have constructed a human hepatocarcinoma HepG2PRDX6-/- cell line using the CRISPR/Cas9 technique and have characterized the cellular response to lack of PRDX6. Applying quantitative global and redox proteomics, flow cytometry, in vivo extracellular flow analysis, Western blot and electron microscopy, we have detected diminished respiratory capacity, downregulation of mitochondrial proteins and altered mitochondrial morphology. Autophagic vesicles were abundant while the unfolded protein response (UPR), HIF1A and NRF2 transcription factors were not activated, despite increased levels of p62/SQSTM1 and reactive oxygen species (ROS). Insulin receptor (INSR), 3-phosphoinositide-dependent protein kinase 1 (PDPK1), uptake of glucose and hexokinase-2 (HK2) decreased markedly while nucleotide biosynthesis, lipogenesis and synthesis of long chain polyunsaturated fatty acids (LC-PUFA) increased. 254 Cys-peptides belonging to 202 proteins underwent significant redox changes. PRDX6 knockout had an antiproliferative effect due to cell cycle arrest at G2/M transition, without signs of apoptosis. Loss of PLA2 may affect the levels of specific lipids altering lipid signaling pathways, while loss of peroxidase activity could induce redox changes at critical sensitive cysteine residues in key proteins. Oxidation of specific cysteines in Proliferating Cell Nuclear Antigen (PCNA) could interfere with entry into mitosis. The GSH/Glutaredoxin system was downregulated likely contributing to these redox changes. Altogether the data demonstrate that loss of PRDX6 slows down cell division and alters metabolism and mitochondrial function, so that cell survival depends on glycolysis to lactate for ATP production and on AMPK-independent autophagy to obtain building blocks for biosynthesis. PRDX6 is an important link in the chain of elements connecting redox homeostasis and proliferation.

Engineered nanomaterials and oxidative stress: current understanding and future challenges.

Engineered nanomaterials (ENMs) are being incorporated at an unprecedented rate into consumer and biomedical products. This increased usage will ultimately lead to increased human exposure; therefore, understanding ENM safety is an important concern to the public. Although ENMs may exert toxicity through multiple mechanisms, one common mechanism of toxicity recognized across a range of ENMs with varying physicochemical properties is oxidative stress. Further, it is recognized that several key physicochemical properties of ENMs including size, material composition, surface chemistry, band gap, and level of ionic dissolution for example contribute to ENM driven oxidative stress. While it has been shown that exposure of cells to ENMs at high acute doses produce reactive oxygen species at a toxic level often leading to cytotoxicity, there is little research looking at oxidative stress caused by ENM exposure at more relevant low or non-toxic doses. Although the former can lead to apoptosis, genotoxicity, and inflammation, the latter can potentially be damaging as chronic changes to the intracellular redox state leads to cellular reprogramming, resulting in disease initiation and progression among other systemic damage. This current opinions article will review the physicochemical properties and mechanisms associated with ENM-driven oxidative stress and will discuss the need for research investigating effects on the redox proteome that may lead to cellular dysfunction at low or chronic doses of ENMs.

Peroxiredoxin 6 Down-Regulation Induces Metabolic Remodeling and Cell Cycle Arrest in HepG2 Cells.

Peroxiredoxin 6 (Prdx6) is the only member of 1-Cys subfamily of peroxiredoxins in human cells. It is the only Prdx acting on phospholipid hydroperoxides possessing two additional sites with phospholipase A2 (PLA2) and lysophosphatidylcholine-acyl transferase (LPCAT) activities. There are contrasting reports on the roles and mechanisms of multifunctional Prdx6 in several pathologies and on its sensitivity to, and influence on, the redox environment. We have down-regulated Prdx6 with specific siRNA in hepatoblastoma HepG2 cells to study its role in cell proliferation, redox homeostasis, and metabolic programming. Cell proliferation and cell number decreased while cell volume increased; import of glucose and nucleotide biosynthesis also diminished while polyamines, phospholipids, and most glycolipids increased. A proteomic quantitative analysis suggested changes in membrane arrangement and vesicle trafficking as well as redox changes in enzymes of carbon and glutathione metabolism, pentose-phosphate pathway, citrate cycle, fatty acid metabolism, biosynthesis of aminoacids, and Glycolysis/Gluconeogenesis. Specific redox changes in Hexokinase-2 (HK2), Prdx6, intracellular chloride ion channel-1 (CLIC1), PEP-carboxykinase-2 (PCK2), and 3-phosphoglycerate dehydrogenase (PHGDH) are compatible with the metabolic remodeling toward a predominant gluconeogenic flow from aminoacids with diversion at 3-phospohglycerate toward serine and other biosynthetic pathways thereon and with cell cycle arrest at G1/S transition.

Toxicant-mediated redox control of proteostasis in neurodegeneration.

Disruption in redox signaling and control of cellular processes has emerged as a key player in many pathologies including neurodegeneration. As protein aggregations are a common hallmark of several neuronal pathologies, a firm understanding of the interplay between redox signaling, oxidative and free radical stress, and proteinopathies is required to sort out the complex mechanisms in these diseases. Fortunately, models of toxicant-induced neurodegeneration can be utilized to evaluate and report mechanistic alterations in the proteostasis network (PN). The epidemiological links between environmental toxicants and neurological disease gives further credence into characterizing the toxicant-mediated PN disruptions observed in these conditions. Reviewed here are examples of mechanistic interaction between oxidative or free radical stress and PN alterations. Additionally, investigations into toxicant-mediated PN disruptions, specifically focusing on environmental metals and pesticides, are discussed. Finally, we emphasize the need to distinguish whether the presence of protein aggregations are contributory to phenotypes related to neurodegeneration, or if they are a byproduct of PN deficiencies.

Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches.

The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto-oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.

Hydrogen Sulfide and Reactive Sulfur Species Impact Proteome S-Sulfhydration and Global Virulence Regulation in Staphylococcus aureus.

Hydrogen sulfide (H2S) is thought to protect bacteria from oxidative stress, but a comprehensive understanding of its function in bacteria is largely unexplored. In this study, we show that the human pathogen Staphylococcus aureus (S. aureus) harbors significant effector molecules of H2S signaling, reactive sulfur species (RSS), as low molecular weight persulfides of bacillithiol, coenzyme A, and cysteine, and significant inorganic polysulfide species. We find that proteome S-sulfhydration, a post-translational modification (PTM) in H2S signaling, is widespread in S. aureus. RSS levels modulate the expression of secreted virulence factors and the cytotoxicity of the secretome, consistent with an S-sulfhydration-dependent inhibition of DNA binding by MgrA, a global virulence regulator. Two previously uncharacterized thioredoxin-like proteins, denoted TrxP and TrxQ, are S-sulfhydrated in sulfide-stressed cells and are capable of reducing protein hydrodisulfides, suggesting that this PTM is potentially regulatory in S. aureus. In conclusion, our results reveal that S. aureus harbors a pool of proteome- and metabolite-derived RSS capable of impacting protein activities and gene regulation and that H2S signaling can be sensed by global regulators to affect the expression of virulence factors.

The cysteine proteome.

The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological switching of structure and reactivity. These biological modifications include sulfenylation and disulfide formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metalation, and other modifications. Extensive knowledge about these systems and their compartmentalization now provides a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed understanding of redox signaling pathways and sensing networks is becoming available to allow the discrimination of network structures. This research focuses attention on the need for atlases of Cys modifications to develop systems biology models. Such atlases will be especially useful for integrative studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to the quantitative proteome in the omics continuum connecting the genome to the exposome.