The physiological functions of supersulfides, inorganic and organic sulfides with sulfur catenation, have been extensively studied. Their synthesis is mainly mediated by mitochondrial cysteinyl-tRNA synthetase (CARS2) that functions as a principal cysteine persulfide synthase. This study aimed to investigate the role of supersulfides in joint homeostasis and bone regeneration. Using Cars2AINK/+ mutant mice, in which the KIIK motif of CARS2 essential for supersulfide production was replaced with AINK, we evaluated the role of supersulfides in fracture healing and cartilage homeostasis during osteoarthritis (OA). Tibial fracture surgery was performed on the wild-type (Cars2+/+) and Cars2AINK/+ mice littermates. Bulk RNA-seq analysis for the osteochondral regeneration in the fracture model showed increased inflammatory markers and reduced osteogenic factors, indicative of impaired bone regeneration, in Cars2AINK/+ mice. Destabilization of the medial meniscus (DMM) surgery was performed to produce the mouse OA model. Histological analyses with Osteoarthritis Research Society International and synovitis scores revealed accelerated OA progression in Cars2AINK/+ mice compared with that in Cars2+/+ mice. To assess the effects of supersulfides on OA progression, glutathione trisulfide (GSSSG) or saline was periodically injected into the mouse knee joints after the DMM surgery. Thus, supersulfides derived from CARS2 and GSSSG exogenously administered significantly inhibited inflammation and lipid peroxidation of the joint cartilage, possibly through suppression of ferroptosis, during OA development. This study represents a significant advancement in understanding anti-inflammatory and anti-oxidant functions of supersulfides in skeletal tissues and may have a clinical relevance for the bone healing and OA therapeutics.
Abstract Here we uncover collagen, the main structural protein of all connective tissues, as a redox‐active material. We identify dihydroxyphenylalanine (DOPA) residues, post‐translational oxidation products of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox‐active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.
Abstract Sulfite oxidase (SOX) deficiency is a rare inborn error of cysteine metabolism resulting in severe neurological damage. In patients, sulfite accumulates to toxic levels causing a raise in downstream products S -sulfocysteine (SSC), mediating excitotoxicity, and thiosulfate, a catabolic intermediate/product of H 2 S metabolism. Here, we report a full-body knock-out mouse model for SOX deficiency (SOXD) with a severely impaired phenotype. Amongst the urinary biomarkers, thiosulfate showed a 45-fold accumulation in SOXD mice representing the major excreted S-metabolite. Consistently, we found increased plasma H 2 S, which was derived from sulfite-induced release from persulfides as demonstrated in vitro and in vivo . Mass spectrometric analysis of total protein persulfidome identified a major loss of persulfidation in 20% of the proteome affecting enzymes in amino acids and fatty acid metabolism. Urinary amino acid profiles indicate metabolic rewiring suggesting partial reversal of the TCA cycle thus identifying a novel contribution of H 2 S metabolism and persulfidation in SOXD.
Abstract 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.
Abstract Here we uncover collagen, the main structural protein of all connective tissues, as a redox-active material. We identify dihydroxyphenylalanine (DOPA) residues, post-translational oxidation products of tyrosine residues, to be common in collagen derived from different connective tissues. We observe that these DOPA residues endow collagen with substantial radical scavenging capacity. When reducing radicals, DOPA residues work as redox relay: they convert to the quinone and generate hydrogen peroxide. In this dual function, DOPA outcompetes its amino acid precursors and ascorbic acid. Our results establish DOPA residues as redox-active side chains of collagens, probably protecting connective tissues against radicals formed under mechanical stress and/or inflammation.
Oxygen molecules accept electrons from the respiratory chain in the mitochondria and are responsible for energy production in aerobic organisms. The reactive oxygen species formed via these oxygen reduction processes undergo complicated electron transfer reactions with other biological substances, which leads to alterations in their physiological functions and cause diverse biological and pathophysiological consequences (e.g., oxidative stress). Oxygen accounts for only a small proportion of the redox reactions in organisms, especially under aerobic or hypoxic conditions but not under anaerobic and hypoxic conditions. This article discusses a completely new concept of redox biology, which is governed by redox-active supersulfides, i.e., sulfur-catenated molecular species. These species are present in abundance in all organisms but remain largely unexplored in terms of redox biology and life science research. In fact, accumulating evidence shows that supersulfides have extensive redox chemical properties and that they can be readily ionized or radicalized to participate in energy metabolism, redox signaling, and oxidative stress responses in cells and in vivo. Thus, pharmacological intervention and medicinal modulation of supersulfide activities have been shown to benefit the regulation of disease pathogenesis as well as disease control.
Initially adopted as a mucolytic about 60 years ago, the cysteine prodrug N-acetylcysteine (NAC) is the standard of care to treat paracetamol intoxication, and is included on the World Health Organization's list of essential medicines. Additionally, NAC increasingly became the epitome of an "antioxidant". Arguably, it is the most widely used "antioxidant" in experimental cell and animal biology, as well as clinical studies. Most investigators use and test NAC with the idea that it prevents or attenuates oxidative stress. Conventionally, it is assumed that NAC acts as (i) a reductant of disulfide bonds, (ii) a scavenger of reactive oxygen species and/or (iii) a precursor for glutathione biosynthesis. While these mechanisms may apply under specific circumstances, they cannot be generalized to explain the effects of NAC in a majority of settings and situations. In most cases the mechanism of action has remained unclear and untested. In this review, we discuss the validity of conventional assumptions and the scope of a newly discovered mechanism of action, namely the conversion of NAC into hydrogen sulfide and sulfane sulfur species. The antioxidative and cytoprotective activities of per- and polysulfides may explain many of the effects that have previously been ascribed to NAC or NAC-derived glutathione.
Protein Modifications. In their Research Article (e202203684), Tobias P. Dick et al. show that persulfide adducts derived from commonly used alkylating agents are unstable under biologically relevant conditions. The adducts convert to thioethers by losing sulfane sulfur to nucleophilic acceptors.
For decades, the major focus of redox biology has been oxygen, the most abundant element on Earth. Molecular oxygen functions as the final electron acceptor in the mitochondrial respiratory chain, contributing to energy production in aerobic organisms. In addition, oxygen-derived reactive oxygen species including hydrogen peroxide and nitrogen free radicals, such as superoxide, hydroxyl radical and nitric oxide radical, undergo a complicated sequence of electron transfer reactions with other biomolecules, which lead to their modified physiological functions and diverse biological and pathophysiological consequences (e.g. oxidative stress). What is now evident is that oxygen accounts for only a small number of redox reactions in organisms and knowledge of biological redox reactions is still quite limited. This article reviews a new aspects of redox biology which is governed by redox-active sulfur-containing molecules-supersulfides. We define the term 'supersulfides' as sulfur species with catenated sulfur atoms. Supersulfides were determined to be abundant in all organisms, but their redox biological properties have remained largely unexplored. In fact, the unique chemical properties of supersulfides permit them to be readily ionized or radicalized, thereby allowing supersulfides to actively participate in redox reactions and antioxidant responses in cells. Accumulating evidence has demonstrated that supersulfides are indispensable for fundamental biological processes such as energy production, nucleic acid metabolism, protein translation and others. Moreover, manipulation of supersulfide levels was beneficial for pathogenesis of various diseases. Thus, supersulfide biology has opened a new era of disease control that includes potential applications to clinical diagnosis, prevention and therapeutics of diseases.
