Protein persulfidation in plants: mechanisms and functions beyond a simple stress response.

Posttranslational modifications (PTMs) can modulate the activity, localization and interactions of proteins and (re)define their biological function. Understanding how changing environments can alter cellular processes thus requires detailed knowledge about the dynamics of PTMs in time and space. A PTM that gained increasing attention in the last decades is protein persulfidation, where a cysteine thiol (-SH) is covalently bound to sulfane sulfur to form a persulfide (-SSH). The precise cellular mechanisms underlying the presumed persulfide signaling in plants are, however, only beginning to emerge. In the mitochondrial matrix, strict regulation of persulfidation and H2S homeostasis is of prime importance for maintaining mitochondrial bioenergetic processes because H2S is a highly potent poison for cytochrome c oxidase. This review summarizes the current knowledge about protein persulfidation and corresponding processes in mitochondria of the model plant Arabidopsis. These processes will be compared to the respective processes in non-plant models to underpin similarities or highlight apparent differences. We provide an overview of mitochondrial pathways that contribute to H2S and protein persulfide generation and mechanisms for H2S fixation and de-persulfidation. Based on current proteomic data, we compile a plant mitochondrial persulfidome and discuss how persulfidation may regulate protein function.

Hydrogen sulfide signaling in plant response to temperature stress.

For the past 300 years, hydrogen sulfide (H2S) has been considered a toxic gas. Nowadays, it has been found to be a novel signaling molecule in plants involved in the regulation of cellular metabolism, seed germination, plant growth, development, and response to environmental stresses, including high temperature (HT) and low temperature (LT). As a signaling molecule, H2S can be actively synthesized and degraded in the cytosol, chloroplasts, and mitochondria of plant cells by enzymatic and non-enzymatic pathways to maintain homeostasis. To date, plant receptors for H2S have not been found. It usually exerts physiological functions through the persulfidation of target proteins. In the past 10 years, H2S signaling in plants has gained much attention. Therefore, in this review, based on that same attention, H2S homeostasis, protein persulfidation, and the signaling role of H2S in plant response to HT and LT stress were summarized. Also, the common mechanisms of H2S-induced HT and LT tolerance in plants were updated. These mechanisms involve restoration of biomembrane integrity, synthesis of stress proteins, enhancement of the antioxidant system and methylglyoxal (MG) detoxification system, improvement of the water homeostasis system, and reestablishment of Ca2+ homeostasis and acid-base balance. These updates lay the foundation for further understanding the physiological functions of H2S and acquiring temperature-stress-resistant crops to develop sustainable food and agriculture.

A role for the cystathionine-ß-synthase /H2S axis in astrocyte dysfunction in the aging brain.

Astrocytic dysfunction is central to age-related neurodegenerative diseases. However, the mechanisms leading to astrocytic dysfunction are not well understood. We identify that among the diverse cellular constituents of the brain, murine and human astrocytes are enriched in the expression of CBS. Depleting CBS in astrocytes causes mitochondrial dysfunction, increases the production of reactive oxygen species (ROS) and decreases cellular bioenergetics that can be partially rescued by exogenous H2S supplementation or by re-expressing CBS. Conversely, the CBS/H2S axis, associated protein persulfidation and proliferation are decreased in astrocytes upon oxidative stress which can be rescued by exogenous H2S supplementation. Here we reveal that in the aging brain, the CBS/H2S axis is downregulated leading to decreased protein persulfidation, together augmenting oxidative stress. Our findings uncover an important protective role of the CBS/H2S axis in astrocytes that may be disrupted in the aged brain.

Photorespiration: regulation and new insights on the potential role of persulfidation.

Photorespiration has been considered a 'futile' cycle in C3 plants, necessary to detoxify and recycle the metabolites generated by the oxygenating activity of Rubisco. However, several reports indicate that this metabolic route plays a fundamental role in plant metabolism and constitutes a very interesting research topic. Many open questions still remain with regard to photorespiration. One of these questions is how the photorespiratory process is regulated in plants and what factors contribute to this regulation. In this review, we summarize recent advances in the regulation of the photorespiratory pathway with a special focus on the transcriptional and post-translational regulation of photorespiration and the interconnections of this process with nitrogen and sulfur metabolism. Recent findings on sulfide signaling and protein persulfidation are also described.

Emerging Chemical Biology of Protein Persulfidation.

Significance: Protein persulfidation (the formation of RSSH), an evolutionarily conserved oxidative posttranslational modification in which thiol groups in cysteine residues are converted into persulfides, has emerged as one of the main mechanisms through which hydrogen sulfide (H2S) conveys its signaling. Recent Advances: New methodological advances in persulfide labeling started unraveling the chemical biology of this modification and its role in (patho)physiology. Some of the key metabolic enzymes are regulated by persulfidation. RSSH levels are important for the cellular defense against oxidative injury, and they decrease with aging, leaving proteins vulnerable to oxidative damage. Persulfidation is dysregulated in many diseases. Critical Issues: A relatively new field of signaling by protein persulfidation still has many unanswered questions: the mechanism(s) of persulfide formation and transpersulfidation and the identification of "protein persulfidases," the improvement of methods to monitor RSSH changes and identify protein targets, and understanding the mechanisms through which this modification controls important (patho)physiological functions. Future Directions: Deep mechanistic studies using more selective and sensitive RSSH labeling techniques will provide high-resolution structural, functional, quantitative, and spatiotemporal information on RSSH dynamics and help with better understanding how H2S-derived protein persulfidation affects protein structure and function in health and disease. This knowledge could pave the way for targeted drug design for a wide variety of pathologies. Antioxid. Redox Signal. 39, 19-39.

Reactive sulfur species and their significance in health and disease.

Reactive sulfur species (RSS) have been recognized in the last two decades as very important molecules in redox regulation. They are involved in metabolic processes and, in this way, they are responsible for maintenance of health. This review summarizes current information about the essential biological RSS, including H2S, low molecular weight persulfides, protein persulfides as well as organic and inorganic polysulfides, their synthesis, catabolism and chemical reactivity. Moreover, the role of RSS disturbances in various pathologies including vascular diseases, chronic kidney diseases, diabetes mellitus Type 2, neurological diseases, obesity, chronic obstructive pulmonary disease and in the most current problem of COVID-19 is presented. The significance of RSS in aging is also mentioned. Finally, the possibilities of using the precursors of various forms of RSS for therapeutic purposes are discussed.

The Combined Partial Knockdown of CBS and MPST Genes Induces Inflammation, Impairs Adipocyte Function-Related Gene Expression and Disrupts Protein Persulfidation in Human Adipocytes.

Recent studies in mice and humans demonstrated the relevance of H2S synthesising enzymes, such as CTH, CBS, and MPST, in the physiology of adipose tissue and the differentiation of preadipocyte into adipocytes. Here, our objective was to investigate the combined role of CTH, CBS, and MPST in the preservation of adipocyte protein persulfidation and adipogenesis. Combined partial CTH, CBS, and MPST gene knockdown was achieved treating fully human adipocytes with siRNAs against these transcripts (siRNA_MIX). Adipocyte protein persulfidation was analyzed using label-free quantitative mass spectrometry coupled with a dimedone-switch method for protein labeling and purification. Proteomic analysis quantified 216 proteins with statistically different levels of persulfidation in KD cells compared to control adipocytes. In fully differentiated adipocytes, CBS and MPST mRNA and protein levels were abundant, while CTH expression was very low. It is noteworthy that siRNA_MIX administration resulted in a significant decrease in CBS and MPST expression, without impacting on CTH. The combined partial knockdown of the CBS and MPST genes resulted in reduced cellular sulfide levels in parallel to decreased expression of relevant genes for adipocyte biology, including adipogenesis, mitochondrial biogenesis, and lipogenesis, but increased proinflammatory- and senescence-related genes. It should be noted that the combined partial knockdown of CBS and MPST genes also led to a significant disruption in the persulfidation pattern of the adipocyte proteins. Although among the less persulfidated proteins, we identified several relevant proteins for adipocyte adipogenesis and function, among the most persulfidated, key mediators of adipocyte inflammation and dysfunction as well as some proteins that might play a positive role in adipogenesis were found. In conclusion, the current study indicates that the combined partial elimination of CBS and MPST (but not CTH) in adipocytes affects the expression of genes related to the maintenance of adipocyte function and promotes inflammation, possibly by altering the pattern of protein persulfidation in these cells, suggesting that these enzymes were required for the functional maintenance of adipocytes.

Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

Protein persulfides (R-S-SH) have emerged as a common post-translational modification. Detection and quantitation of protein persulfides requires trapping with alkylating agents. Here we show that alkylating agents differ dramatically in their ability to conserve the persulfide's sulfur-sulfur bond for subsequent detection by mass spectrometry. The two alkylating agents most commonly used in cell biology and biochemistry, N-ethylmaleimide and iodoacetamide, are found to be unsuitable for the purpose of conserving persulfides under biologically relevant conditions. The resulting persulfide adducts (R-S-S-Alk) rapidly convert into the corresponding thioethers (R-S-Alk) by donating sulfur to ambient nucleophilic acceptors. In contrast, certain other alkylating agents, in particular monobromobimane and N-t-butyl-iodoacetamide, generate stable alkylated persulfides. We propose that the nature of the alkylating agent determines the ability of the disulfide bond (R-S-S-Alk) to tautomerize into the thiosulfoxide (R-(S=S)-Alk), and/or the ability of nucleophiles to remove the sulfane sulfur atom from the thiosulfoxide.

Sulfane Sulfur Regulates LasR-Mediated Quorum Sensing and Virulence in Pseudomonas aeruginosa PAO1.

Sulfane sulfur, such as inorganic and organic polysulfide (HSn- and RSn-, n > 2), is a common cellular component, produced either from hydrogen sulfide oxidation or cysteine metabolism. In Pseudomonas aeruginosa PAO1, LasR is a quorum sensing master regulator. After binding its autoinducer, LasR binds to its target DNA to activate the transcription of a suite of genes, including virulence factors. Herein, we report that the production of hydrogen sulfide and sulfane sulfur were positively correlated in P. aeruginosa PAO1, and sulfane sulfur was able to modify LasR, which generated Cys188 persulfide and trisulfide and produced a pentasulfur link between Cys201 and Cys203. The modifications did not affect LasR binding to its target DNA site, but made it several-fold more effective than unmodified LasR in activating transcription in both in vitro and in vivo assays. On the contrary, H2O2 inactivates LasR via producing a disulfide bond between Cys201 and Cys203. P. aeruginosa PAO1 had a high cellular sulfane sulfur and high LasR activity in the mid log phase and early stationary phase, but a low sulfane sulfur and low LasR activity in the declination phase. Thus, sulfane sulfur is a new signaling factor in the bacterium, adding another level of control over LasR-mediated quorum sensing and turning down the activity in old cells.

Proteomics Profiling of S-sulfurated Proteins in Acinetobacter baumannii.

Hydrogen sulfide (H2S) is emerging as an important modulator in bacterial cytoprotection against the host immune response in infected animals, which may well be attributed to downstream highly oxidized sulfur species, termed reactive sulfur species (RSS), derived from H2S. One mechanism by which H2S/RSS may signal in the cell is through proteome S-sulfuration (persulfidation), which is the conversion of protein thiols (-SH) to protein persulfides (-SSH). While several analytical methods have been developed to profile sites of protein persulfidation, few have been applied to bacterial cells. The analytical workflow presented here was recently utilized to profile proteome persulfidation in the major human pathogen Acinetobacter baumannii treated with an exogenous sulfide source, Na2S. The data obtained using this protocol allow quantitation of the change in persulfidation status of each cysteine in the proteome normalized to the change in protein abundance, thus identifying sites of persulfidation that may constitute regulatory modifications. These can be validated using follow-up biochemical studies.

Activation of Endogenous H2S Biosynthesis or Supplementation with Exogenous H2S Enhances Adipose Tissue Adipogenesis and Preserves Adipocyte Physiology in Humans.

Aims: To investigate the impact of exogenous hydrogen sulfide (H2S) and its endogenous biosynthesis on human adipocytes and adipose tissue in the context of obesity and insulin resistance. Results: Experiments in human adipose tissue explants and in isolated preadipocytes demonstrated that exogenous H2S or the activation of endogenous H2S biosynthesis resulted in increased adipogenesis, insulin action, sirtuin deacetylase, and PPARγ transcriptional activity, whereas chemical inhibition and gene knockdown of each enzyme generating H2S (CTH, CBS, MPST) led to altered adipocyte differentiation, cellular senescence, and increased inflammation. In agreement with these experimental data, visceral and subcutaneous adipose tissue expression of H2S-synthesising enzymes was significantly reduced in morbidly obese subjects in association with attenuated adipogenesis and increased markers of adipose tissue inflammation and senescence. Interestingly, weight-loss interventions (including bariatric surgery or diet/exercise) improved the expression of H2S biosynthesis-related genes. In human preadipocytes, the expression of CTH, CBS, and MPST genes and H2S production were dramatically increased during adipocyte differentiation. More importantly, the adipocyte proteome exhibiting persulfidation was characterized, disclosing that different proteins involved in fatty acid and lipid metabolism, the citrate cycle, insulin signaling, several adipokines, and PPAR, experienced the most dramatic persulfidation (85-98%). Innovation: No previous studies investigated the impact of H2S on human adipose tissue. This study suggests that the potentiation of adipose tissue H2S biosynthesis is a possible therapeutic approach to improve adipose tissue dysfunction in patients with obesity and insulin resistance. Conclusion: Altogether, these data supported the relevance of H2S biosynthesis in the modulation of human adipocyte physiology. Antioxid. Redox Signal. 35, 319-340.

The multifaceted roles of sulfane sulfur species in cancer-associated processes.

Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (H2S)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that H2S, currently recognized as a key signaling molecule in human physiology and pathophysiology, plays an important role in cancer biology by modulating cell bioenergetics and contributing to metabolic reprogramming. This is accomplished through functional modulation of target proteins via H2S binding to heme iron centers or H2S-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of H2S but also as key mediators of some of the biological effects commonly attributed to H2S, the multifaceted role of these species in cancer biology is reviewed here with reference to H2S, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties.

Opposing effects of polysulfides and thioredoxin on apoptosis through caspase persulfidation.

Hydrogen sulfide has been implicated in a large number of physiological processes including cell survival and death, encouraging research into its mechanisms of action and therapeutic potential. Results from recent studies suggest that the cellular effects of hydrogen sulfide are mediated in part by sulfane sulfur species, including persulfides and polysulfides. In the present study, we investigated the apoptosis-modulating effects of polysulfides, especially on the caspase cascade, which mediates the intrinsic apoptotic pathway. Biochemical analyses revealed that organic or synthetic polysulfides strongly and rapidly inhibit the enzymatic activity of caspase-3, a major effector protease in apoptosis. We attributed the caspase-3 inhibition to persulfidation of its catalytic cysteine. In apoptotically stimulated HeLa cells, short-term exposure to polysulfides triggered the persulfidation and deactivation of cleaved caspase-3. These effects were antagonized by the thioredoxin/thioredoxin reductase system (Trx/TrxR). Trx/TrxR restored the activity of polysulfide-inactivated caspase-3 in vitro, and TrxR inhibition potentiated polysulfide-mediated suppression of caspase-3 activity in situ We further found that under conditions of low TrxR activity, early cell exposure to polysulfides leads to enhanced persulfidation of initiator caspase-9 and decreases apoptosis. Notably, we show that the proenzymes procaspase-3 and -9 are basally persulfidated in resting (unstimulated) cells and become depersulfidated during their processing and activation. Inhibition of TrxR attenuated the depersulfidation and activation of caspase-9. Taken together, our results reveal that polysulfides target the caspase-9/3 cascade and thereby suppress cancer cell apoptosis, and highlight the role of Trx/TrxR-mediated depersulfidation in enabling caspase activation.

Selective Persulfide Detection Reveals Evolutionarily Conserved Antiaging Effects of S-Sulfhydration.

Life on Earth emerged in a hydrogen sulfide (H2S)-rich environment eons ago and with it protein persulfidation mediated by H2S evolved as a signaling mechanism. Protein persulfidation (S-sulfhydration) is a post-translational modification of reactive cysteine residues, which modulate protein structure and/or function. Persulfides are difficult to label and study due to their reactivity and similarity with cysteine. Here, we report a facile strategy for chemoselective persulfide bioconjugation using dimedone-based probes, to achieve highly selective, rapid, and robust persulfide labeling in biological samples with broad utility. Using this method, we show persulfidation is an evolutionarily conserved modification and waves of persulfidation are employed by cells to resolve sulfenylation and prevent irreversible cysteine overoxidation preserving protein function. We report an age-associated decline in persulfidation that is conserved across evolutionary boundaries. Accordingly, dietary or pharmacological interventions to increase persulfidation associate with increased longevity and improved capacity to cope with stress stimuli.

Hydrogen sulfide modulates eukaryotic translation initiation factor 2α (eIF2α) phosphorylation status in the integrated stress-response pathway.

Hydrogen sulfide (H2S) regulates various physiological processes, including neuronal activity, vascular tone, inflammation, and energy metabolism. Moreover, H2S elicits cytoprotective effects against stressors in various cellular models of injury. However, the mechanism of the signaling pathways mediating the cytoprotective functions of H2S is not well understood. We previously uncovered a heme-dependent metabolic switch for transient induction of H2S production in the trans-sulfuration pathway. Here, we demonstrate that increased endogenous H2S production or its exogenous administration modulates major components of the integrated stress response promoting a metabolic state primed for stress response. We show that H2S transiently increases phosphorylation of eukaryotic translation initiation factor 2 (eIF2α) resulting in inhibition of general protein synthesis. The H2S-induced increase in eIF2α phosphorylation was mediated at least in part by inhibition of protein phosphatase-1 (PP1c) via persulfidation at Cys-127. Overexpression of a PP1c cysteine mutant (C127S-PP1c) abrogated the H2S effect on eIF2α phosphorylation. Our data support a model in which H2S exerts its cytoprotective effect on ISR signaling by inducing a transient adaptive reprogramming of global mRNA translation. Although a transient increase in endogenous H2S production provides cytoprotection, its chronic increase such as in cystathionine ß-synthase deficiency may pose a problem.