Unlocking Selective Anticancer Mechanisms: Dinuclear Manganese Superoxide Dismutase Mimetics Combined with Pt(II) Complexes.

We exploration the in vitro activities of the dinuclear Mn2L2Ac and Mn2L2 complexes (where HL = 2-{[di(2-pyridyl)methylamino]-methyl}phenol), possessing dual superoxide dismutase (SOD) and catalase (CAT) activity, both individually and in conjunction with various Pt(II)-complexes, either as mixtures or as the Mn2-Pt adducts. Our findings revealed a notable up to 50% enhancement in the viability of healthy human breast cells, contrasted with a viability decrease as low as 50% in breast cancer cells upon combined treatments with Mn2 SOD mimics and Pt(II) complexes. Specifically, we synthesized and characterized the self-assembled Mn2-Pt adducts (isolated Mn2L2Pt and in situ Mn2L2Pt'), linking Mn2L2-core with the carboxylate group of PtDAPCl2 (dichloro(2,3-diaminopropionic acid) platinum(II)). The SOD activity of the isolated Mn2L2Pt adduct (kSOD = 1.7 × 107 M-1 s-1) remained intact. We elucidated key mechanisms underlying the observed biological effects. We demonstrated that Mn2-containing formulations predominantly target mitochondrial processes, differently affecting the proteome of cancerous and healthy cells. They induced downregulation of H2S signaling and expression of mitochondrial complex I and III, as well as increased oxidative phosphorylation pathways and upregulation of EGFR in cancer cells. In contrast, healthy cells showed a decrease in EGFR expression and a moderate enrichment in oxidative phosphorylation pathways.

Fungal and host protein persulfidation are functionally correlated and modulate both virulence and antifungal response.

Aspergillus fumigatus is a human fungal pathogen that can cause devastating pulmonary infections, termed "aspergilloses," in individuals suffering immune imbalances or underlying lung conditions. As rapid adaptation to stress is crucial for the outcome of the host-pathogen interplay, here we investigated the role of the versatile posttranslational modification (PTM) persulfidation for both fungal virulence and antifungal host defense. We show that an A. fumigatus mutant with low persulfidation levels is more susceptible to host-mediated killing and displays reduced virulence in murine models of infection. Additionally, we found that a single nucleotide polymorphism (SNP) in the human gene encoding cystathionine γ-lyase (CTH) causes a reduction in cellular persulfidation and correlates with a predisposition of hematopoietic stem cell transplant recipients to invasive pulmonary aspergillosis (IPA), as correct levels of persulfidation are required for optimal antifungal activity of recipients' lung resident host cells. Importantly, the levels of host persulfidation determine the levels of fungal persulfidation, ultimately reflecting a host-pathogen functional correlation and highlighting a potential new therapeutic target for the treatment of aspergillosis.

Hydrogen sulfide is neuroprotective in Alzheimer's disease by sulfhydrating GSK3ß and inhibiting Tau hyperphosphorylation.

Alzheimer's disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFTs), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFTs, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H2S) is dysregulated during aging. H2S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine γ-lyase (CSE), the biosynthetic enzyme for H2S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H2S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3ß (GSK3ß). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H2S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD.

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.

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.

Oxidation of methionine residues activates the high-threshold heat-sensitive ion channel TRPV2.

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.

Nitroxyl Delivered by Angeli's Salt Causes Short-Lasting Activation Followed by Long-Lasting Deactivation of Meningeal Afferents in Models of Headache Generation.

The role of TRPA1 receptor channels in meningeal nociception underlying the generation of headaches is still unclear. Activating as well as inhibitory effects of TRPA1 agonists have been reported in animal models of headache. The aim of the present study was to clarify the effect of the TRPA1 agonist nitroxyl (HNO) delivered by Angeli's salt in two rodent models of meningeal nociception. Single fibre recordings were performed using half-skull preparations of mice (C57BL/6) in vitro. Angeli's salt solution (AS, 300 µM) caused short-lasting vigorous increases in neuronal activity of primary meningeal afferents, followed by deactivation and desensitisation. These effects were similar in TRPA1 knockout and even more pronounced in TRPA1/TRPV1 double-knockout mice in comparison to wild-type mice. The activity of spinal trigeminal neurons with afferent input from the dura mater was recorded in vivo in anesthetised rats. AS (300 µM) or the TRPA1 agonist acrolein (100 and 300 µM) was applied to the exposed dura mater. AS caused no significant changes in spontaneous activity, while the mechanically evoked activity was reduced after acrolein application. These results do not confirm the assumption that activation of trigeminal TRPA1 receptor channels triggers the generation of headaches or contributes to its aggravation. Instead, there is evidence that TRPA1 activation may have an inhibitory function in the nociceptive trigeminal system.

Targeting Zinc Finger Proteins with Exogenous Metals and Molecules: Lessons learned from Tristetraprolin, a CCCH type Zinc Finger.

ZF proteins are ubiquitous eukaryotic proteins that play important roles in gene regulation. ZFs contain small domains made up of a combination of four cysteine and histidine residues, and are classified based up on the identity of these residues and their spacing. One emerging class of ZFs are the Cys3His (or CCCH) class of ZFs. These ZFs play key roles in regulating RNA. In this minireview, an overview of the CCCH class of ZFs, with a focus on tristetraprolin (TTP) is provided. TTP regulates inflammation by controlling cytokine mRNAs, and there is an interest in modulating TTP activity to control inflammation. Two methods to control TTP activity are to target with exogenous metals (a 'metals in medicine' approach) or to target with endogenous signaling molecules. Work that has been done to target TTP with Fe, Cu, Cd and Au as well as with H2S is reviewed. This includes attention to new methods that have been developed to monitor metal exchange with the spectroscopically silent ZnII including native electro-spray ionization mass spectrometry (ESI-MS), spin-filter inductively coupled plasma mass spectrometry (ICP-MS) and cryo-electro-spray mass spectrometry (CSI-MS); along with fluorescence anisotropy (FA) to follow RNA binding.

Chemical Biology of H2S Signaling through Persulfidation.

Signaling by H2S is proposed to occur via persulfidation, a posttranslational modification of cysteine residues (RSH) to persulfides (RSSH). Persulfidation provides a framework for understanding the physiological and pharmacological effects of H2S. Due to the inherent instability of persulfides, their chemistry is understudied. In this review, we discuss the biologically relevant chemistry of H2S and the enzymatic routes for its production and oxidation. We cover the chemical biology of persulfides and the chemical probes for detecting them. We conclude by discussing the roles ascribed to protein persulfidation in cell signaling pathways.

Electrophile-Induced Conformational Switch of the Human TRPA1 Ion Channel Detected by Mass Spectrometry.

The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Δ1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1.

Saying NO to H2S: A Story of HNO, HSNO, and SSNO.

Interactions between small inorganic molecules are fundamental to the understanding of basic reaction mechanisms and some of the initial processes of chemical evolution that preceded organic molecules and led to the origin of life. The kinetics of these processes are suitable for the fast generation of a variety of new chemical entities and the propagation of a cascade of chemical reactions, a property that is ideal for signaling purposes even in biological systems. NO and H2S are such molecules that are nowadays recognized as biological gasotransmitters involved in the regulation of physiological functions through protein modifications such as S-nitrosothiol, disulfide, and persulfide formations. In this Viewpoint, we review the current understanding of interactions of NO (and organic and metal nitrosyl species) with H2S, in both chemical and biochemical contexts. Through the formation of HNO, (H)SNO (and its isomers), (H)SSNO, and polysulfides, these two gasotransmitters initiate reaction networks with significant roles in cell signaling. The chemical reactivities and biological effects of these nitrogen and sulfur species are still unresolved, and, thus, a cross-talk between all of them represents a challenging interdisciplinary field that awaits exciting new findings. We tackle some of the intriguing and open questions and provide perspectives for future research directions.

Direct Zinc Finger Protein Persulfidation by H2 S Is Facilitated by Zn2.

H2 S is a gaseous signaling molecule that modifies cysteine residues in proteins to form persulfides (P-SSH). One family of proteins modified by H2 S are zinc finger (ZF) proteins, which contain multiple zinc-coordinating cysteine residues. Herein, we report the reactivity of H2 S with a ZF protein called tristetraprolin (TTP). Rapid persulfidation leading to complete thiol oxidation of TTP mediated by H2 S was observed by low-temperature ESI-MS and fluorescence spectroscopy. Persulfidation of TTP required O2 , which reacts with H2 S to form superoxide, as detected by ESI-MS, a hydroethidine fluorescence assay, and EPR spin trapping. H2 S was observed to inhibit TTP function (binding to TNFα mRNA) by an in vitro fluorescence anisotropy assay and to modulate TNFα in vivo. H2 S was unreactive towards TTP when the protein was bound to RNA, thus suggesting a protective effect of RNA.

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin.

Hydrogen sulfide is a critical signaling molecule, but high concentrations cause cellular toxicity. A four-enzyme pathway in the mitochondrion detoxifies H2S by converting it to thiosulfate and sulfate. Recent studies have shown that globins like hemoglobin and myoglobin can also oxidize H2S to thiosulfate and hydropolysulfides. Neuroglobin, a globin enriched in the brain, was reported to bind H2S tightly and was postulated to play a role in modulating neuronal sensitivity to H2S in conditions such as stroke. However, the H2S reactivity of the coordinately saturated heme in neuroglobin is expected a priori to be substantially lower than that of the 5-coordinate hemes present in myoglobin and hemoglobin. To resolve this discrepancy, we explored the role of the distal histidine residue in muting the reactivity of human neuroglobin toward H2S. Ferric neuroglobin is slowly reduced by H2S and catalyzes its inefficient oxidative conversion to thiosulfate. Mutation of the distal His64 residue to alanine promotes rapid binding of H2S and its efficient conversion to oxidized products. X-ray absorption, EPR, and resonance Raman spectroscopy highlight the chemically different reaction options influenced by the distal histidine ligand. This study provides mechanistic insights into how the distal heme ligand in neuroglobin caps its reactivity toward H2S and identifies by cryo-mass spectrometry a range of sulfide oxidation products with 2-6 catenated sulfur atoms with or without oxygen insertion, which accumulate in the absence of the His64 ligand.

Assessment of H2S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA.

Hydrogen sulfide (H2S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H2S source and sink, and many of the biological effects of H2S relate to its interactions with mitochondria. However, the significance of mitochondrial H2S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo Although a number of fluorescent H2S probes have been developed these are best suited to cells in culture and cannot be used in vivo To address this unmet need we have developed a mitochondria-targeted H2S probe, MitoA, which can be used to assess relative changes in mitochondrial H2S levels in vivo MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues in vivo There, the aryl azido group reacts with H2S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H2S in vivo Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial H2S levels in vivo As a proof of principle we used MitoA to show that H2S levels increase in vivo during myocardial ischemia.

Switching between Inner- and Outer-Sphere PCET Mechanisms of Small-Molecule Activation: Superoxide Dismutation and Oxygen/Superoxide Reduction Reactivity Deriving from the Same Manganese Complex.

Readily exchangeable water molecules are commonly found in the active sites of oxidoreductases, yet the overwhelming majority of studies on small-molecule mimics of these enzymes entirely ignores the contribution of water to the reactivity. Studies of how these enzymes can continue to function in spite of the presence of highly oxidizing species are likewise limited. The mononuclear MnII complex with the potentially hexadentate ligand N-(2-hydroxy-5-methylbenzyl)-N,N',N'-tris(2-pyridinylmethyl)-1,2-ethanediamine (LOH) was previously found to act as both a H2O2-responsive MRI contrast agent and a mimic of superoxide dismutase (SOD). Here, we studied this complex in aqueous solutions at different pH values in order to determine its (i) acid-base equilibria, (ii) coordination equilibria, (iii) substitution lability and operative mechanisms for water exchange, (iv) redox behavior and ability to participate in proton-coupled electron transfer (PCET) reactions, (v) SOD activity and reductive activity toward both oxygen and superoxide, and (vi) mechanism for its transformation into the binuclear MnII complex with (H)OL-LOH and its hydroxylated derivatives. The conclusions drawn from potentiometric titrations, low-temperature mass spectrometry, temperature- and pressure-dependent 17O NMR spectroscopy, electrochemistry, stopped-flow kinetic analyses, and EPR measurements were supported by the structural characterization and quantum chemical analysis of proposed intermediate species. These comprehensive studies enabled us to determine how transiently bound water molecules impact the rate and mechanism of SOD catalysis. Metal-bound water molecules facilitate the PCET necessary for outer-sphere SOD activity. The absence of the water ligand, conversely, enables the inner-sphere reduction of both superoxide and dioxygen. The LOH complex maintains its SOD activity in the presence of •OH and MnIV-oxo species by channeling these oxidants toward the synthesis of a functionally equivalent binuclear MnII species.

Cellular Fates of Manganese(II) Pentaazamacrocyclic Superoxide Dismutase (SOD) Mimetics: Fluorescently Labeled MnSOD Mimetics, X-ray Absorption Spectroscopy, and X-ray Fluorescence Microscopy Studies.

Manganese(II) pentaazamacrocyclic complexes (MnPAMs) can act as small-molecule mimics of manganese superoxide dismutase (MnSOD) with potential therapeutic application in conditions linked to oxidative stress. Previously, the in vitro mechanism of action has been determined, their activity has been demonstrated in cells, and some representatives of this class of MnSOD mimetics have entered clinical trials. However, MnPAM uptake, distribution, and metabolism in cells are largely unknown. Therefore, we have used X-ray fluorescence microscopy (XFM) and X-ray absorption spectroscopy (XAS) to study the cellular fate of a number of MnPAMs. We have also synthesized and characterized fluorescently labeled (pyrene and rhodamine) manganese(II) pyane [manganese(II) trans-2,13-dimethyl-3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene] derivatives and investigated their utility for cellular imaging of MnPAMs. Their SOD activity was determined via a direct stopped-flow technique. XFM experiments show that treatment with amine-based manganese(II) pyane type pentaazamacrocycles leads to a 10-100-fold increase in the overall cellular manganese levels compared to the physiological levels of manganese in control cells. In treated cells in general, manganese was distributed throughout the cell body, with a couple of notable exceptions. The lipophilicity of the MnPAMs, examined by partitioning in octanol-buffer system, was a good predictor of the relative cellular manganese levels. Analysis of the XAS data of treated cells revealed that some fraction of amine-based MnPAMs taken up by the cells remained intact, with the rest transformed into SOD-active manganese(II) phosphate. Higher phosphate binding constants, determined from the effect of the phosphate concentration on in vitro SOD activity, were associated with more extensive metabolism of the amine-based MnPAMs to manganese(II) phosphate. In contrast, the imine-based manganese(II) pydiene complex that is prone to hydrolysis was entirely decomposed after uptake and free manganese(II) was oxidized to a manganese(III) oxide type species, in cytosolic compartments, possibly mitochondria. Complex stability constants (determined for some of the MnPAMs) are less indicative of the cellular fate of the complexes than the corresponding phosphate binding constants.

Reaction of Hydrogen Sulfide with Disulfide and Sulfenic Acid to Form the Strongly Nucleophilic Persulfide.

Hydrogen sulfide (H2S) is increasingly recognized to modulate physiological processes in mammals through mechanisms that are currently under scrutiny. H2S is not able to react with reduced thiols (RSH). However, H2S, more precisely HS(-), is able to react with oxidized thiol derivatives. We performed a systematic study of the reactivity of HS(-) toward symmetric low molecular weight disulfides (RSSR) and mixed albumin (HSA) disulfides. Correlations with thiol acidity and computational modeling showed that the reaction occurs through a concerted mechanism. Comparison with analogous reactions of thiolates indicated that the intrinsic reactivity of HS(-) is 1 order of magnitude lower than that of thiolates. In addition, H2S is able to react with sulfenic acids (RSOH). The rate constant of the reaction of H2S with the sulfenic acid formed in HSA was determined. Both reactions of H2S with disulfides and sulfenic acids yield persulfides (RSSH), recently identified post-translational modifications. The formation of this derivative in HSA was determined, and the rate constants of its reactions with a reporter disulfide and with peroxynitrite revealed that persulfides are better nucleophiles than thiols, which is consistent with the α effect. Experiments with cells in culture showed that treatment with hydrogen peroxide enhanced the formation of persulfides. Biological implications are discussed. Our results give light on the mechanisms of persulfide formation and provide quantitative evidence for the high nucleophilicity of these novel derivatives, setting the stage for understanding the contribution of the reactions of H2S with oxidized thiol derivatives to H2S effector processes.

Streptozotocin Stimulates the Ion Channel TRPA1 Directly: INVOLVEMENT OF PEROXYNITRITE.

Streptozotocin (STZ)-induced diabetes is the most commonly used animal model of diabetes. Here, we have demonstrated that intraplantar injections of low dose STZ evoked acute polymodal hypersensitivities in mice. These hypersensitivities were inhibited by a TRPA1 antagonist and were absent in TRPA1-null mice. In wild type mice, systemic STZ treatment (180 mg/kg) evoked a loss of cold and mechanical sensitivity within an hour of injection, which lasted for at least 10 days. In contrast, Trpa1(-/-) mice developed mechanical, cold, and heat hypersensitivity 24 h after STZ. The TRPA1-dependent sensory loss produced by STZ occurs before the onset of diabetes and may thus not be readily distinguished from the similar sensory abnormalities produced by the ensuing diabetic neuropathy. In vitro, STZ activated TRPA1 in isolated sensory neurons, TRPA1 cell lines, and membrane patches. Mass spectrometry studies revealed that STZ oxidizes TRPA1 cysteines to disulfides and sulfenic acids. Furthermore, incubation of tyrosine with STZ resulted in formation of dityrosine, suggesting formation of peroxynitrite. Functional analysis of TRPA1 mutants showed that cysteine residues that were oxidized by STZ were important for TRPA1 responsiveness to STZ. Our results have identified oxidation of TRPA1 cysteine residues, most likely by peroxynitrite, as a novel mechanism of action of STZ. Direct stimulation of TRPA1 complicates the interpretation of results from STZ models of diabetic sensory neuropathy and strongly argues that more refined models of diabetic neuropathy should replace the use of STZ.

Biosynthesis and Reactivity of Cysteine Persulfides in Signaling.

Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys-SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys-SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys-SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys-SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys-SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys-SSH product is short and decomposition leads to a mixture of polysulfides (Cys-S-(S)n-S-Cys). These in vitro data, together with the intrinsic reactivity of Cys-SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.

Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.