Hydrogen sulfide-mediated persulfidation regulates homocysteine metabolism and enhances ferroptosis in non-small cell lung cancer.

Hydrogen sulfide (H2S), a metabolite of the transsulfuration pathway, has been implicated in ferroptosis, a unique form of cell death caused by lipid peroxidation. While the exact mechanisms controlling ferroptosis remain unclear, our study reveals that H2S sensitizes human non-small cell lung cancer (NSCLC) cells to this process, particularly when cysteine levels are low. Combining H2S with cystine depletion significantly enhances the effectiveness of ferroptosis-based cancer therapy. Mechanistically, H2S persulfidates the 195th cysteine on S-adenosyl homocysteine hydrolase (SAHH), reducing its enzymatic activity. This leads to decreased homocysteine levels, subsequently lowering cysteine and glutathione concentrations under cystine depletion conditions. These changes ultimately increase the vulnerability of NSCLC cells to ferroptosis. Our findings establish H2S as a key regulator of homocysteine metabolism and a critical factor in determining NSCLC cell susceptibility to ferroptosis. These results highlight the potential of H2S-based therapies to improve the efficacy of ferroptosis-targeted cancer treatments for NSCLC.

E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to cysteine persulfidation.

Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Urm1 has also been observed to conjugate to target proteins like other UBLs, the molecular mechanism of its attachment remains unknown. Here, we reconstitute the covalent attachment of thiocarboxylated Urm1 to various cellular target proteins in vitro, revealing that, unlike other known UBLs, this process is E2/E3-independent and requires oxidative stress. Furthermore, we present the crystal structures of the peroxiredoxin Ahp1 before and after the covalent attachment of Urm1. Surprisingly, we show that urmylation is accompanied by the transfer of sulfur to cysteine residues in the target proteins, also known as cysteine persulfidation. Our results illustrate the role of the Uba4-Urm1 system as a key evolutionary link between prokaryotic SCPs and the UBL modifications observed in modern eukaryotes.

The Prognostic Biomarker RAB7A Promotes Growth and Metastasis of Liver Cancer Cells by Regulating Glycolysis and YAP1 Activation.

Activating transcription factor 6 (ATF6) and its downstream genes are involved in progression of hepatocellular carcinoma (HCC). Herein, we demonstrated that sulfhydration of Ras-related protein Rab-7a (RAB7A) was regulated by ATF6. High expression of RAB7A indicated poor prognosis of HCC patients. RAB7A overexpression contributed to proliferation, colony formation, migration, and invasion of HepG2 and Hep3B cells. Furthermore, we found that RAB7A enhanced aerobic glycolysis in HepG2 cells, indicating a higher degree of tumor malignancy. Mechanistically, RAB7A suppressed Yes-associated protein 1 (YAP1) binding to 14-3-3 and conduced to YAP1 nuclear translocation and activation, promoting its downstream gene expression, thereby promoting growth and metastasis of liver cancer cells. In addition, knocking down RAB7A attenuated the progression of orthotopic liver tumors in mice. These findings illustrate the important role of RAB7A in regulating HCC progression. Thus, RAB7A may be a potential innovative target for HCC treatment.

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 antagonizes cytokinin to change root system architecture through persulfidation of CKX2 in Arabidopsis.

Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule, which has been shown to play an important role in plant growth and development by coupling with various phytohormones. However, the relationship between H2S and cytokinin (CTK) and the mechanisms by which H2S and CTK affect root growth remain poorly understood. Endogenous CTK was analyzed by UHPLC-ESI-MS/MS. Persulfidation of cytokinin oxidase/dehydrogenases (CKXs) was analyzed by mass spectrometry (MS). ckx2/CKX2wild-type (WT), OE CKX2 and ckx2/CKX2Cys(C)62alanine(A) transgenic lines were isolated with the ckx2 background. H2S is linked to CTK content by CKX2, which regulates root system architecture (RSA). Persulfidation at cysteine (Cys)62 residue of CKX2 enhances CKX2 activity, resulting in reduced CTK content. We utilized 35S-LCD/oasa1 transgenic lines to investigate the effect of endogenous H2S on RSA, indicating that H2S reduces the gravitropic set-point angle (GSA), shortens root hairs, and increases the number of lateral roots (LRs). The persulfidation of CKX2Cys62 changes the elongation of cells on the upper and lower flanks of LR elongation zone, confirming that Cys62 of CKX2 is the specificity target of H2S to regulate RSA in vivo. In conclusion, this study demonstrated that H2S negatively regulates CTK content and affects RSA by persulfidation of CKX2Cys62 in Arabidopsis thaliana.

Ascorbate peroxidase in fruits and modulation of its activity by reactive species.

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate-glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source. APX is present in all photosynthetic eukaryotes from algae to higher plants and, at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including the apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. APX activity can be modulated by various post-translational modifications including tyrosine nitration, S-nitrosation, persulfidation, and S-sulfenylation. This allows the connection of H2O2 metabolism with other relevant signaling molecules such as NO and H2S, thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process and during post-harvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and, more recently, melatonin is seen as a new alternative to maintain and extend the shelf life and quality of fruits because they can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered as new biotechnological tools to improve crop quality in the horticultural industry.

Hydrogen sulfide supplementation as a potential treatment for primary mitochondrial diseases.

Primary mitochondrial diseases (PMD) are amongst the most common inborn errors of metabolism causing fatal outcomes within the first decade of life. With marked heterogeneity in both inheritance patterns and physiological manifestations, these conditions present distinct challenges for targeted drug therapy, where effective therapeutic countermeasures remain elusive within the clinic. Hydrogen sulfide (H2S)-based therapeutics may offer a new option for patient treatment, having been proposed as a conserved mitochondrial substrate and post-translational regulator across species, displaying therapeutic effects in age-related mitochondrial dysfunction and neurodegenerative models of mitochondrial disease. H2S can stimulate mitochondrial respiration at sites downstream of common PMD-defective subunits, augmenting energy production, mitochondrial function and reducing cell death. Here, we highlight the primary signalling mechanisms of H2S in mitochondria relevant for PMD and outline key cytoprotective proteins/pathways amenable to post-translational restoration via H2S-mediated persulfidation. The mechanisms proposed here, combined with the advent of potent mitochondria-targeted sulfide delivery molecules, could provide a framework for H2S as a countermeasure for PMD disease progression.

Persulfidation of ATG18a regulates autophagy under ER stress in Arabidopsis.

Hydrogen sulfide (H2S) is an endogenously generated gaseous signaling molecule, which recently has been implicated in autophagy regulation in both plants and mammals through persulfidation of specific targets. Persulfidation has been suggested as the molecular mechanism through which sulfide regulates autophagy in plant cells. ATG18a is a core autophagy component that is required for bulk autophagy and also for reticulophagy during endoplasmic reticulum (ER) stress. In this research, we revealed the role of sulfide in plant ER stress responses as a negative regulator of autophagy. We demonstrate that sulfide regulates ATG18a phospholipid-binding activity by reversible persulfidation at Cys103, and that this modification activates ATG18a binding capacity to specific phospholipids in a reversible manner. Our findings strongly suggest that persulfidation of ATG18a at C103 regulates autophagy under ER stress, and that the impairment of persulfidation affects both the number and size of autophagosomes.

Insights from Structure-Based Simulations into the Persulfidation of Uridine Diphosphate-Glycosyltransferase71c5 Facilitating the Reversible Inactivation of Abscisic Acid.

The action of abscisic acid (ABA) is closely related to its level in plant tissues. Uridine diphosphate-glycosyltransferase71c5 (UGT71C5) was characterized as a major UGT enzyme to catalyze the formation of the ABA-glucose ester (ABA-GE), a reversible inactive form of free ABA in Arabidopsis thaliana (thale cress). UGTs function in a mode where the catalytic base deprotonates an acceptor to allow a nucleophilic attack at the anomeric center of the donor, achieving the transfer of a glucose moiety. The proteomic data revealed that UGT71C5 can be persulfidated. Herein, an experimental method was employed to detect the persulfidation site of UGT71C5, and the computational methods were further used to identify the yet unknown molecular basis of ABA glycosylation as well as the regulatory role of persulfidation in this process. Our results suggest that the linker and the U-shaped loop are regulatory structural elements: the linker is associated with the binding of uridine diphosphate glucose (UPG) and the U-shaped loop is involved in binding both UPG and ABA.It was also found that it is through tuning the dynamics of the U-shaped loop that is accompanied by the movement of tyrosine (Y388) that the persulfidation of cysteine (C311) leads to the catalytic residue histidine (H16) being in place, preparing for the deprotonation of ABA, and then reorientates UPG and deprotonated ABA closer to the 'Michaelis' complex, facilitating the transfer of a glucose moiety. Ultimately, the persulfidation of UGT71C5 is in favor of ABA glycosylation. Our results provide insights into the molecular details of UGT71C5 recognizing substrates and insights concerning persulfidation as a possible mechanism for hydrogen sulfide (H2S) to modulate the content of ABA, which helps us understand how modulating ABA level strengthens plant tolerance.

NO and H2S Contribute to Crop Resilience against Atmospheric Stressors.

Atmospheric stressors include a variety of pollutant gases such as CO2, nitrous oxide (NOx), and sulfurous compounds which could have a natural origin or be generated by uncontrolled human activity. Nevertheless, other atmospheric elements including high and low temperatures, ozone (O3), UV-B radiation, or acid rain among others can affect, at different levels, a large number of plant species, particularly those of agronomic interest. Paradoxically, both nitric oxide (NO) and hydrogen sulfide (H2S), until recently were considered toxic since they are part of the polluting gases; however, at present, these molecules are part of the mechanism of response to multiple stresses since they exert signaling functions which usually have an associated stimulation of the enzymatic and non-enzymatic antioxidant systems. At present, these gasotransmitters are considered essential components of the defense against a wide range of environmental stresses including atmospheric ones. This review aims to provide an updated vision of the endogenous metabolism of NO and H2S in plant cells and to deepen how the exogenous application of these compounds can contribute to crop resilience, particularly, against atmospheric stressors stimulating antioxidant systems.

Hydrogen Sulfide in the Oxidative Stress Response of Plants: Crosstalk with Reactive Oxygen Species.

Growing evidence suggests that exposure of plants to unfavorable environments leads to the accumulation of hydrogen sulfide (H2S) and reactive oxygen species (ROS). H2S interacts with the ROS-mediated oxidative stress response network at multiple levels. Therefore, it is essential to elucidate the mechanisms by which H2S and ROS interact. The molecular mechanism of action by H2S relies on the post-translational modification of the cysteine sulfur group (-SH), known as persulfidation. H2S cannot react directly with -SH, but it can react with oxidized cysteine residues, and this oxidation process is induced by H2O2. Evidently, ROS is involved in the signaling pathway of H2S and plays a significant role. In this review, we summarize the role of H2S-mediated post-translational modification mechanisms in oxidative stress responses. Moreover, the mechanism of interaction between H2S and ROS in the regulation of redox reactions is focused upon, and the positive cooperative role of H2S and ROS is elucidated. Subsequently, based on the existing evidence and clues, we propose some potential problems and new clues to be explored, which are crucial for the development of the crosstalk mechanism of H2S and ROS in plants.

Chemoselective Proteomics, Zinc Fingers, and a Zinc(II) Model for H2S Mediated Persulfidation.

The gasotransmitter hydrogen sulfide (H2S) is thought to be involved in the post-translational modification of cysteine residues to produce reactive persulfides. A persulfide-specific chemoselective proteomics approach with mammalian cells has identified a broad range of zinc finger (ZF) proteins as targets of persulfidation. Parallel studies with isolated ZFs show that persulfidation is mediated by ZnII, O2, and H2S, with intermediates involving oxygen- and sulfur-based radicals detected by mass spectrometry and optical spectroscopies. A small molecule ZnII complex exhibits analogous reactivity with H2S and O2, giving a persulfidated product. These data show that ZnII is not just a biological structural element, but also plays a critical role in mediating H2S-dependent persulfidation. ZF persulfidation appears to be a general post-translational modification and a possible conduit for H2S signaling. This work has implications for our understanding of H2S-mediated signaling and the regulation of ZFs in cellular physiology and development.

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.

Persulfidation protects from oxidative stress under nonphotorespiratory conditions in Arabidopsis.

Hydrogen sulfide is a signaling molecule in plants that regulates essential biological processes through protein persulfidation. However, little is known about sulfide-mediated regulation in relation to photorespiration. Here, we performed label-free quantitative proteomic analysis and observed a high impact on protein persulfidation levels when plants grown under nonphotorespiratory conditions were transferred to air, with 98.7% of the identified proteins being more persulfidated under suppressed photorespiration. Interestingly, a higher level of reactive oxygen species (ROS) was detected under nonphotorespiratory conditions. Analysis of the effect of sulfide on aspects associated with non- or photorespiratory growth conditions has demonstrated that it protects plants grown under suppressed photorespiration. Thus, sulfide amends the imbalance of carbon/nitrogen and restores ATP levels to concentrations like those of air-grown plants; balances the high level of ROS in plants under nonphotorespiratory conditions to reach a cellular redox state similar to that in air-grown plants; and regulates stomatal closure, to decrease the high guard cell ROS levels and induce stomatal aperture. In this way, sulfide signals the CO2 -dependent stomata movement, in the opposite direction of the established abscisic acid-dependent movement. Our findings suggest that the high persulfidation level under suppressed photorespiration reveals an essential role of sulfide signaling under these conditions.

Persulfidation maintains cytosolic G6PDs activity through changing tetrameric structure and competing cysteine sulfur oxidation under salt stress in Arabidopsis and tomato.

Glucose-6-phosphate dehydrogenases (G6PDs) are essential regulators of cellular redox. Hydrogen sulfide (H2 S) is a small gasotransmitter that improves plant adaptation to stress; however, its role in regulating G6PD oligomerization to resist oxidative stress remains unknown in plants. Persulfidation of cytosolic G6PDs was analyzed by mass spectrometry (MS). The structural change model of AtG6PD6 homooligomer was built by chemical cross-linking coupled with mass spectrometry (CXMS). We isolated AtG6PD6C159A and SlG6PDCC155A transgenic lines to confirm the in vivo function of persulfidated sites with the g6pd5,6 background. Persulfidation occurs at Arabidopsis G6PD6 Cystine (Cys)159 and tomato G6PDC Cys155, leading to alterations of spatial distance between lysine (K)491-K475 from 42.0 Å to 10.3 Å within the G6PD tetramer. The structural alteration occurs in the structural NADP+ binding domain, which governs the stability of G6PD homooligomer. Persulfidation enhances G6PD oligomerization, thereby increasing substrate affinity. Under high salt stress, cytosolic G6PDs activity was inhibited due to oxidative modifications. Persulfidation protects these specific sites and prevents oxidative damage. In summary, H2 S-mediated persulfidation promotes cytosolic G6PD activity by altering homotetrameric structure. The cytosolic G6PD adaptive regulation with two kinds of protein modifications at the atomic and molecular levels is critical for the cellular stress response.

Persulfidation of plant and bacteroid proteins is involved in legume nodule development and senescence.

Legumes establish symbiosis with rhizobia forming nitrogen-fixing nodules. The central role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in nodule biology has been clearly established. Recently, hydrogen sulfide (H2S) and other reactive sulfur species (RSS) have emerged as novel signaling molecules in animals and plants. A major mechanism by which ROS, RNS, and RSS fulfil their signaling role is the post-translational modification of proteins. To identify possible functions of H2S in nodule development and senescence, we used the tag-switch method to quantify changes in the persulfidation profile of common bean (Phaseolus vulgaris) nodules at different developmental stages. Proteomic analyses indicate that persulfidation plays a regulatory role in plant and bacteroid metabolism and senescence. The effect of a H2S donor on nodule functioning and on several proteins involved in ROS and RNS homeostasis was also investigated. Our results using recombinant proteins and nodulated plants support a crosstalk among H2S, ROS and RNS, a protective function of persulfidation on redox-sensitive enzymes, and a beneficial effect of H2S on symbiotic nitrogen fixation. We conclude that the general decrease of persulfidation levels observed in plant proteins of aging nodules is one of the mechanisms that disrupt redox homeostasis leading to senescence.

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.

Transcriptional and post-translational regulation of plant autophagy.

In response to changing environmental conditions, plants activate cellular responses to enable them to adapt. One such response is autophagy, in which cellular components, for example proteins and organelles, are delivered to the vacuole for degradation. Autophagy is activated by a wide range of conditions, and the regulatory pathways controlling this activation are now being elucidated. However, key aspects of how these factors may function together to properly modulate autophagy in response to specific internal or external signals are yet to be discovered. In this review we discuss mechanisms for regulation of autophagy in response to environmental stress and disruptions in cell homeostasis. These pathways include post-translational modification of proteins required for autophagy activation and progression, control of protein stability of the autophagy machinery, and transcriptional regulation, resulting in changes in transcription of genes involved in autophagy. In particular, we highlight potential connections between the roles of key regulators and explore gaps in research, the filling of which can further our understanding of the autophagy regulatory network in plants.

Sulfide promotes tolerance to drought through protein persulfidation in Arabidopsis.

Hydrogen sulfide (H2S) is a signaling molecule that regulates essential plant processes. In this study, the role of H2S during drought was analysed, focusing on the underlying mechanism. Pretreatments with H2S before imposing drought on plants substantially improved the characteristic stressed phenotypes under drought and decreased the levels of typical biochemical stress markers such as anthocyanin, proline, and hydrogen peroxide. H2S also regulated drought-responsive genes and amino acid metabolism, and repressed drought-induced bulk autophagy and protein ubiquitination, demonstrating the protective effects of H2S pretreatment. Quantitative proteomic analysis identified 887 significantly different persulfidated proteins between control and drought stress plants. Bioinformatic analyses of the proteins more persulfidated in drought revealed that the most enriched biological processes were cellular response to oxidative stress and hydrogen peroxide catabolism. Protein degradation, abiotic stress responses, and the phenylpropanoid pathway were also highlighted, suggesting the importance of persulfidation in coping with drought-induced stress. Our findings emphasize the role of H2S as a promoter of enhanced tolerance to drought, enabling plants to respond more rapidly and efficiently. Furthermore, the main role of protein persulfidation in alleviating reactive oxygen species accumulation and balancing redox homeostasis under drought stress is highlighted.

Hydrogen sulfide improves salt tolerance through persulfidation of PMA1 in Arabidopsis.

KEY MESSAGE: A new interaction was found between PMA1 and GRF4. H2S promotes the interaction through persulfidated Cys446 of PMA1. H2S activates PMA1 to maintain K+/Na+ homeostasis through persulfidation under salt stress. Plasma membrane H+-ATPase (PMA) is a transmembrane transporter responsible for pumping protons, and its contribution to salt resistance is indispensable in plants. Hydrogen sulfide (H2S), a small signaling gas molecule, plays the important roles in facilitating adaptation of plants to salt stress. However, how H2S regulates PMA activity remains largely unclear. Here, we show a possible original mechanism for H2S to regulate PMA activity. PMA1, a predominant member in the PMA family of Arabidopsis, has a non-conservative persulfidated cysteine (Cys) residue (Cys446), which is exposed on the surface of PMA1 and located in cation transporter/ATPase domain. A new interaction of PMA1 and GENERAL REGULATORY FACTOR 4 (GRF4, belongs to the 14-3-3 protein family) was found by chemical crosslinking coupled with mass spectrometry (CXMS) in vivo. H2S-mediated persulfidation promoted the binding of PMA1 to GRF4. Further studies showed that H2S enhanced instantaneous H+ efflux and maintained K+/Na+ homeostasis under salt stress. In light of these findings, we suggest that H2S promotes the binding of PMA1 to GRF4 through persulfidation, and then activating PMA, thus improving the salt tolerance of Arabidopsis.