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Selenium (Se) is an essential trace element for life. Seleno-methylselenocysteine (SeMCys) can serve as a Se supplement with anticarcinogenic activity and can improve cognitive deficits. We engineered Escherichia coli for microbial production of SeMCys. The genes involved in the synthesis of SeMCys were divided into three modules–the selenocysteine (SeCys) synthesis, methyl donor synthesis and SMT modules–and expressed in plasmids with different copy numbers. The higher copy number of the SeCys synthesis module facilitated SeMCys production. The major routes for SeCys degradation were then modified. Deletion of the cysteine desulfurase gene csdA or sufS improved SeMCys production the most, and the strain that knocked out both genes doubled SeMCys production. The addition of serine in the mid-logarithmic growth phase significantly improved SeMCys synthesis. When the serine synthetic pathway was enhanced, SeMCys production increased by 12.5 %. Fed-batch culture for sodium selenite supplementation in the early stationary phase improved SeMCys production to 3.715 mg/L. This is the first report of the metabolic engineering of E. coli for the production of SeMCys and provide information on Se metabolism. • Higher copy number of the SeCys synthesis module facilitated SeMCys production. • Cysteine desulfurase CsdA and SufS are involved in the degradation of SeCys. • The enhanced Ser synthetic pathway increased SeMCys synthesis. [ABSTRACT FROM AUTHOR]

• It emphasizes the critical importance of novel therapeutic approaches. • It is the first to discusses the functions of L-cysteine in the anxiety. • It provides an overview of the basic features of L-cysteine. • It presents potential mechanism of L-cysteine in the treatment of anxiety. • It reviews the pathogenesis and neuroanatomical basis of anxiety disorders. Anxiety disorders are prevalent chronic psychological disease with complex pathogenic mechanisms. Current anxiolytics have limited efficacy and numerous side effects in many anxiety patients, highlighting the urgent need for new therapies. Recent research has been focusing on nutritional supplements, particularly amino acids, as potential therapies for anxiety disorders. Among these, L-Cysteine plays a crucial role in various biological processes. L-Cysteine exhibits antioxidant properties that can enhance the antioxidant functions of the central nervous system (CNS). Furthermore, metabolites of L-cysteine, such as glutathione and hydrogen sulfide have been shown to alleviate anxiety through distinct molecular mechanisms. Long-term administration of L-Cysteine has anxiolytic, antidepressant, and memory-improving effects. L-Cysteine depletion can lead to increased oxidative stress in the brain. This review delves into the potential mechanisms of L-Cysteine and its main products, glutathione (GSH) and hydrogen sulfide (H2S) in the management of anxiety and related diseases. [ABSTRACT FROM AUTHOR]

Fe-S clusters are critical cofactors for redox chemistry in all organisms. The cysteine desulfurase, SufS, provides sulfur in the SUF Fe-S cluster bioassembly pathway. SufS is a dimeric, pyridoxal 5'-phosphate-dependent enzyme that uses cysteine as a substrate to generate alanine and a covalent persulfide on an active site cysteine residue. SufS enzymes are activated by an accessory transpersulfurase protein, either SufE or SufU depending on the organism, which accepts the persulfide product and delivers it to downstream partners for Fe-S assembly. Here, using Escherichia coli proteins, we present the first X-ray crystal structure of a SufS/SufE complex. There is a 1:1 stoichiometry with each monomeric unit of the EcSufS dimer bound to one EcSufE subunit, though one EcSufE is rotated ∼7° closer to the EcSufS active site. EcSufE makes clear interactions with the α16 helix of EcSufS and site-directed mutants of several α16 residues were deficient in EcSufE binding. Analysis of the EcSufE structure showed a loss of electron density at the EcSufS/EcSufE interface for a flexible loop containing the highly conserved residue R119. An R119A EcSufE variant binds EcSufS but is not active in cysteine desulfurase assays and fails to support Fe-S cluster bioassembly in vivo. 35S-transfer assays suggest that R119A EcSufE can receive a persulfide, suggesting the residue may function in a release mechanism. The structure of the EcSufS/EcSufE complex allows for comparison with other cysteine desulfurases to understand mechanisms of protected persulfide transfer across protein interfaces. [ABSTRACT FROM AUTHOR]

Reformulation with addition of antioxidants is one potential mitigation strategy to prevent or reduce nitrosamine drug substance-related impurities (NDSRIs) in drug products. To explore whether there could be other approaches to demonstrate bioequivalence for a reformulated oral product, which typically needs in vivo bioequivalence studies to support the changes after approval, the effects of antioxidant on the in vitro permeability of BCS III model drug substances were investigated to see whether there could be any potential impact on drug absorption. Six antioxidants were screened and four (ascorbic acid, cysteine, α-tocopherol and propyl gallate) were selected based on their nitrosamine inhibition efficiencies. The study demonstrated that these four antioxidants, at the tested amounts, did not have observable impact on the in vitro permeability of the BCS III model drug substances across Caco-2 cell monolayers in the In Vitro Dissolution Absorption System (IDAS). An in vitro permeability study could be considered as part of one potential bioequivalence bridging approach for reformulated low-risk immediate release solid oral products and oral suspension products. Other factors such as the influence of antioxidants on intestinal transporter activities should be considered where appropriate. [ABSTRACT FROM AUTHOR]

l -cysteine, a primary building block of mycothiol, plays an essential role in the defense mechanism of Mycobacterium tuberculosis (Mtb). However, it is unclear how Mtb regulates cysteine biosynthesis as no study has reported the cysteine regulatory complex (CRC) in Mtb. Serine acetyltransferase (SAT) and cysteine synthase (CS) interact to form CRC. Although Mt CS has been characterized well, minimal information is available on Mt SAT, which synthesizes, O-acetylserine (OAS), the precursor of cysteine. This study fills the gap and provides experimental evidence for the presence of Mt CRC and a non-canonical multi-oligomeric Mt SAT. We employed multiple analytical methods to characterize the oligomeric and kinetic properties of Mt SAT and Mt CRC. Results show that Mt SAT, lacking >75 N-terminal amino acids exists in three different assembly states; trimer, hexamer, and dodecamer, compared to the single hexameric state of SAT of other bacteria. While hexamers display the highest catalytic turnover, the trimer is the least active. The predominance of trimers at low physiologically relevant concentrations suggests that Mt SAT displays the lowest catalytic potential known. Further, the catalytic potential of Mt SAT is also significantly reduced in CRC state, in contrast to enhanced activity of SAT in CRC of other organisms. Our study provides insights into multi-oligomeric Mt SAT with reduced catalytic potential and demonstrates that both Mt SAT and Mt CS of Mycobacterium interact to form CRC, although with altered catalytic properties. We discuss our results in light of the altered biochemistry of the last step of canonical sulfate-dependent cysteine biosynthesis of Mycobacterium. [Display omitted] • Serine acetyltransferase from Mycobacterium tuberculosis (Mt SAT) lacks four N-terminal helices. • Mt SAT exists in three states, with the trimer demonstrating the least catalytic efficiency. • Only hexamers can form cysteine regulatory complex (CRC) with cysteine synthase, not trimers. • Together, results indicate that compromised catalytic efficiency of Mt SAT may be an evolutionary adaptation. [ABSTRACT FROM AUTHOR]

Non-alcoholic steatohepatitis (NASH), now known as metabolic dysfunction-associated steatohepatitis (MASH), involves oxidative stress caused by the overproduction of reactive oxygen species (ROS). Small-molecule antioxidants have not been approved for antioxidant chemotherapy because of severe adverse effects that collapse redox homeostasis, even in healthy tissues. To overcome these disadvantages, we have been developing poly(ethylene glycol)- block -poly(cysteine) (PEG- block -PCys)-based self-assembling polymer nanoparticles (NanoCyses), releasing Cys after in vivo degradation by endogenous enzymes, to obtain antioxidant effects without adverse effects. However, a comprehensive investigation of the effects of polymer design on therapeutic outcomes has not yet been conducted to develop our NanoCys system for antioxidant chemotherapy. In this study, we synthesized different poly(L -cysteine) (PCys) chains whose sulfanyl groups were protected by tert -butyl thiol (S t Bu) and butyryl (Bu) groups to change the reactivity of the side chains, affording NanoCys(SS) and NanoCys(Bu), respectively. To elucidate the importance of the polymer design, these NanoCyses were orally administered to MASH model mice as a model of oxidative stress-related diseases. Consequently, the acyl-protective NanoCys(Bu) significantly suppressed hepatic lipid accumulation and oxidative stress compared to NanoCys(SS). Furthermore, we substantiated that shorter PCys were much better than longer PCys for therapeutic outcomes and the effects related to the liberation properties of Cys from these nanoparticles. Owing to its antioxidant functions, NanoCyses also significantly attenuated hepatic inflammation and fibrosis in the MASH mouse model. [Display omitted] [ABSTRACT FROM AUTHOR]

The enzymatically regioselective catalyzed incorporation of cysteine sulfoxide into histidine generates physiologically important antioxidants such as ergothioneine and ovothiol. In this issue of Structure , Ireland et al. 1 report the crystal structure of EgtB-IV, which provides insights into the convergent evolution of sulfoxide synthase. The enzymatically regioselective catalyzed incorporation of cysteine sulfoxide into histidine generates physiologically important antioxidants such as ergothioneine and ovothiol. In this issue of Structure , Ireland et al. report the crystal structure of EgtB-IV, which provides insights into the convergent evolution of sulfoxide synthase. [ABSTRACT FROM AUTHOR]

Drug conjugation to an antibody can affect its stability, which depends on factors such as the conjugation technique used, drug-linker properties, and stress encountered. This study focused on the effects of agitation stress on the physical stability of two lysine (ADC-K) and two interchain cysteine (ADC-C) conjugates of an IgG 1 monoclonal antibody (mAb) linked to either ∼4 MMAE or DM1 payloads. During agitation, all antibody–drug conjugates (ADCs) exhibited higher aggregation than the mAb, which was dependent on the conjugation technique (aggregation of ADC-Ks > ADC-Cs) and drug-linker (aggregation of ADCs with MMAE > ADCs with DM1). The aggregation propensities correlated well with higher self-interaction, hydrophobicity, and surface activity of ADCs relative to the mAb. The intermediate reduced mAb (mAb-SH) showed even higher aggregation than the final product ADC-Cs. However, blocking mAb-SH's free thiols with N-ethylmaleimide (NEM) strongly reduced its aggregation, suggesting that free thiols should be minimized in cysteine ADCs. Further, this study demonstrates that a low-volume surface tension method can be used for estimating agitation-induced aggregation of ADCs in early development phases. Identifying liabilities to agitation stress and their relationship to biophysical properties may help optimize ADC stability. [ABSTRACT FROM AUTHOR]

Breeding-induced endometritis is a physiological reaction to clear the uterus from excess spermatozoa and bacteria after breeding. Cysteine rich secretory protein 3 in seminal plasma (spCRISP3) protects spermatozoa from binding and destruction by uterine PMNs, but it is not clear if this involves all sperm and bacteria, or if it is selective to a sub-population of live sperm. The objective of this report was to determine if spCRISP3 (1) is selective in its suppression of PMN-binding to sperm based on viability of spermatozoa, (2) protects bacteria from binding to PMNs, and (3) to determine the localization pattern of spCRISP3 on viable and dead sperm. Semen was collected from five stallions and each ejaculate was divided into (1) live and (2) snap frozen (dead) sperm. Two distinct sperm populations were confirmed by DNA fragmentation and membrane integrity assays. CRISP3 was purified from pooled seminal plasma, and binding of PMNs (isolated from peripheral blood) to the two sperm populations and E. coli was evaluated with flow cytometry in the presence of spCRISP3. In addition, localization of spCRISP3 on live and dead spermatozoa was determined by immunocytochemistry. Comparisons between treatments were analyzed using a one-way-ANOVA and Bonferroni's comparison test, or Kruskal-Wallis ANOVA if not normally distributed. spCRISP3 significantly suppressed binding of PMNs to live spermatozoa (p < 0.0001) but had no effect on dead sperm or bacteria (p > 0.05). Immunocytochemistry confirmed binding of spCRISP3 to live, but not dead spermatozoa. It was concluded that a selective interaction between spCRISP3 and live spermatozoa may be part of a biological mechanism that allows safe transport of viable spermatozoa to the oviducts, while enabling dead spermatozoa and bacteria to be eliminated in a timely fashion after breeding. • Equine CRISP3 binds selectively to live spermatozoa. • CRISP3 in equine seminal plasma suppresses PMN-binding to live, but not dead spermatozoa. • Equine CRISP3 had no effect of PMN-binding to bacteria. • SpCRISP3 appears to be involved in selective sperm transport and elimination from the equine uterus. [ABSTRACT FROM AUTHOR]

Understanding the unique metabolic pathway of L. donovani is crucial for comprehending its biology under oxidative stress conditions. The de novo cysteine biosynthetic pathway of L. donovani is absent in humans and its product, cysteine regulates the downstream components of trypanothione-based thiol metabolism, important for maintaining cellular redox homeostasis. The role of serine o -acetyl transferase (SAT), the first enzyme of this pathway remains unexplored. In order to investigate the role of SAT protein, we cloned SAT gene into pXG-GFP+ vector for episomal expression of SAT in Amphotericin B sensitive L. donovani promastigotes. The SAT overexpression was confirmed by SAT enzymatic assay, GFP fluorescence, immunoblotting and PCR. Our study unveiled an upregulated expression of both LdSAT and LdCS of cysteine biosynthetic pathway and other downstream thiol pathway proteins in LdSAT-OE promastigotes. Additionally, there was an increase in enzymatic activities of LdSAT and LdCS proteins in LdSAT-OE, which was found similar to the Amp B resistant parasites, indicating a potential role of SAT protein in modulating drug resistance. We observed that the overexpression of SAT in Amp B sensitive parasites increases tolerance to drug pressure and oxidative stress via trypanothione-dependent antioxidant mechanism. Moreover, the in vitro J774A.1 macrophage infectivity assessment showed that SAT overexpression augments parasite infectivity. In LdSAT-OE promastigotes, antioxidant enzyme activities like APx and SOD were upregulated, intracellular reactive oxygen species were reduced with a corresponding increase in thiol level, emphasizing SAT's role in stress tolerance and enhanced infectivity. Additionally, the ROS mediated upregulation in the expression of LdSAT, LdCS, LdTryS and LdcTXNPx proteins reveals an essential cross talk between SAT and proteins of thiol metabolism in combating oxidative stress and maintaining redox homeostasis. Taken together, our results provide the first insight into the role of SAT protein in parasite infectivity and survival under drug pressure and oxidative stress. [Display omitted] • SAT is involved in trypanothione-based redox metabolism. • SAT overexpression increases tolerance to oxidative stress. • SAT overexpression confers resistance to Amp B and miltefosine. • SAT overexpression augments antioxidant activities of APx and SOD. • SAT overexpression enhances parasite infectivity and survival under oxidative stress conditions. [ABSTRACT FROM AUTHOR]

Hypothermic liquid storage at 4–5 °C has emerged as a novel approach for preserving boar semen, offering innovative possibilities for semen preservation. However, this method also presents challenges, including cold shock and excessive reactive oxygen species (ROS) production. Therefore, reducing oxidative damage induced by low temperatures becomes essential while supplementing appropriate protectants. In this study, we investigated the efficacy of Bovine Serum Albumin (BSA) compared to Polyvinylpyrrolidone (PVP) and Skim Milk Powder (SMP) in maintaining boar sperm motility and progressive motility using computer-assisted sperm analysis (CASA). Among the tested concentrations, 4 g/L of BSA exhibited the best protective effect. Subsequently, we supplemented different concentrations of l -cysteine (LC) and N-acetyl- l -cysteine (NAC) as additives in the presence of BSA as a protectant. Our results demonstrated that 1 mmol/L of LC and 0.5 mmol/L of NAC exhibited superior protection of sperm quality compared to other concentrations. Furthermore, the 1 mmol/L LC and 0.5 mmol/L NAC groups showed significantly improved plasma membrane integrity and acrosome integrity compared to the control group. These groups also exhibited enhanced antioxidant capacity, evidenced by increased mitochondrial membrane potential (MMP), ATP production, total superoxide dismutase (T-SOD) activity, total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), and GPX-4 levels. Additionally, they demonstrated decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as reduced oxidized glutathione (GSSG) and glutathione reductase (GR) levels. Furthermore, LC and NAC treatment enhanced AMP-activated protein kinase (AMPK) phosphorylation. However, inhibiting AMPK using compound C did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. These findings suggest that 4 g/L BSA can serve as an effective protectant for hypothermic liquid storage of boar semen. Additionally, LC and NAC supplementation reduces oxidative damage by enhancing antioxidant capacity rather than through AMPK-mediated ATP supplementation. These results contribute to advancing the application of LC and NAC in hypothermic liquid storage of boar semen. • 4 g/L BSA can serve as an effective cryoprotectant for hypothermic liquid storage of boar semen. • LC and NAC supplementation reduces oxidative damage through enhancing antioxidant capacity. • Inhibiting AMPK did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. [ABSTRACT FROM AUTHOR]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and 'moonlighting' activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H 2 O 2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H 2 O 2 - and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation. [Display omitted] • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an oxidant-sensitive cysteine (Cys) in its active site. • Effects of macromolecular crowding on GAPDH oxidation by H 2 O 2 and SIN-1 was investigated. • Dextran differentially affects H 2 O 2 - and SIN-1-mediated GAPDH cross-linking. • Higher yields of active site Cys-derived sulfonic acids were detected with crowding. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) is a risk factor for developing active tuberculosis (TB) with a 3-fold increase in susceptibility and a 4-fold higher relapse rate. With increasing DM prevalence in TB endemic regions, understanding pathophysiological changes associated with DM-TB comorbidity is imperative. In this study, streptozotocin (STZ)-induced DM C57BL/6 mice were aerosol infected with low dose (100-120 CFU) Mycobacterium tuberculosis H37Rv. At 3 weeks post infection (w.p.i.), multiple tissue mycobacterial load and metabolites were profiled. The liver proteome of DM-TB and controls were analyzed using quantitative proteomics, and multi-omics data were integrated. DM-TB mice showed dysregulated multi-tissue (lungs, liver, brain, kidney and thigh muscle) metabolism. In contrast, the mycobacterial burden in the lung, spleen and liver was similar at 3 w.p.i. in DM-TB and TB groups. Enrichment analysis of deregulated liver metabolites (n = 20; log2DM-TB/ TB>±1.0) showed significant perturbation in cysteine-methionine, glycine-serine, BCAA and fatty acid metabolism. 60 out of 1660 identified liver proteins showed deregulation (log2DM-TB/TB>±1.0) and contributed from perturbed cysteine-methionine metabolism corroborating metabolomics data. In addition, amino acid biosynthesis, retinol metabolism and polyol biosynthetic process were also differentially enriched in the livers of DM-TB groups. Global correlation analysis of liver metabolome and proteome data showed a strong association between aspartic acid, pyruvic acid, leucine and isoleucine with CYP450 enzymes involved in retinol metabolism, while iminodiacetic acid, isoleucine and γ-aminobutyric acid (GABA) strong positive correlation involved in cysteine metabolism. Targeting perturbed cysteine metabolism using micro molecules, like DL-Propargylglycine, might help prevent liver damage in DM-TB comorbid conditions. [ABSTRACT FROM AUTHOR]

Oxidative stress is inherent to bacterial survival in a range of environments. Recently, a role for cysteine in bacterial defence against oxidative stress has begun to emerge. In a variety of pathogenic and nonpathogenic bacteria, cysteine has been linked to other aspects of the bacterial lifestyle associated with the ability to resist oxidative stress, including virulence and antibiotic resistance. The capacity of bacteria to generate the antioxidant H 2 S from cysteine plays an additional role in the defence against oxidative stress. Interestingly, there appear to be subtle distinctions in the mechanism and role of cysteine in different bacterial species. Further and more thorough analyses into the role of intracellular cysteine regulation in a broader range of bacterial species hold promise in enhancing the understanding of this important system. Reactive oxygen species (ROS), including the superoxide radical anion (O 2 •–), hydrogen peroxide (H 2 O 2), and the hydroxyl radical (•HO), are inherent components of bacterial metabolism in an aerobic environment. Bacteria also encounter exogenous ROS, such as those produced by the host cells during the respiratory burst. As ROS have the capacity to damage bacterial DNA, proteins, and lipids, detoxification of ROS is critical for bacterial survival. It has been recently recognised that low-molecular-weight (LMW) thiols play a central role in this process. Here, we review the emerging role of cysteine in bacterial resistance to ROS with a link to broader elements of bacterial lifestyle closely associated with cysteine-mediated oxidative stress response, including virulence and antibiotic resistance. [ABSTRACT FROM AUTHOR]

d -Amino acids (d- AAs) form a novel class of vital signaling molecules that control brain homeostasis. Abnormal d -AAs levels in biofluids and tissues occur in various brain disorders. d -AAs can be used as biomarkers for disease diagnosis, prognosis, and treatment monitoring. d -AA signaling and metabolic pathways are promising druggable targets for chronic and acute neuropathologies. Free d -amino acids (d -AAs) are emerging as a novel and important class of signaling molecules in many organs, including the brain and endocrine systems. There has been considerable progress in our understanding of the fundamental roles of these atypical messengers, with increasingly recognized implications in a wide range of neuropathologies, including schizophrenia (SCZ), epilepsy, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), substance abuse, and chronic pain, among others. Research has enabled the discovery that d -serine, d -aspartate and more recently d -cysteine are essential for the healthy development and function of the central nervous system (CNS). We discuss recent progress that has profoundly transformed our vision of numerous physiological processes but has also shown how d -AAs are now offering therapeutic promise in clinical settings for several human diseases. [ABSTRACT FROM AUTHOR]

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygendependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERFVIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress. [ABSTRACT FROM AUTHOR]

Group VII ethylene-responsive factor (ERFVII) transcription factors are crucial for the adaption of plants to conditions that limit oxygen availability. A recent study by Zubrycka et al. reveals new aspects of ERFVII stabilization through the PLANT CYSTEINE OXIDASE (PCO)-N degron pathway and non-autonomous regulation in response to different endogenous and exogenous cues. [ABSTRACT FROM AUTHOR]

The available collection of warheads has been extended to target a wider range of amino acid residues beyond cysteine enabling the identification of a larger pool of proteins. Covalent labeling can be achieved by noncatalytic chemical reactions; however, catalytic labeling achieved by heterogenous catalysts, photo- or electrocatalysis is an emerging field increasing the toolkit of specific labeling reactions. Photo- and electrocatalysis provide additional options for conditional labeling. Covalent fragments allow target identification by chemoproteomic techniques and enable identifying new (allosteric) binding sites. Covalent fragments can be considered as viable chemical starting points of targeted covalent ligands for target validation, molecular glues, and proteolysis-targeting chimeras (PROTACs). Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimeras (PROTACs), and their utility in chemical proteomics-based target identification and validation. Covalent fragment approaches combine advantages of covalent binders and fragment-based drug discovery (FBDD) for target identification and validation. Although early applications focused mostly on cysteine labeling, the chemistries of available warheads that target other orthosteric and allosteric protein nucleophiles has recently been extended. The range of different warheads and labeling chemistries provide unique opportunities for screening and optimizing warheads necessary for targeting non-cysteine residues. In this review, we discuss these recently developed amino-acid-specific and promiscuous warheads, as well as emerging labeling chemistries, which includes novel transition metal catalyzed, photoactive, electroactive, and noncatalytic methodologies. We also highlight recent applications of covalent fragments for the development of molecular glues and proteolysis-targeting chimaeras (PROTACs), and their utility in chemical proteomics-based target identification and validation. [ABSTRACT FROM AUTHOR]

The cryopreservation process is associated with the generation of excessive reactive oxygen species, which causes a series of cellular damage to spermatozoa. The objective of the current study was to investigate the effect of different concentrations of cysteine on post-thaw sperm quality of brown-marbled grouper sperm. Semen samples were frozen with cysteine supplemented at 0.5, 1, 2, 5, 10 mM and the control group (no additive). After thawing, sperm quality parameters were analyzed. In comparison to the control, cysteine treatment groups yielded relatively higher sperm total motility, progressive motility, and curvilinear velocity. Different concentrations of cysteine had no effect on average path velocity, straight linear velocity and viability (P > 0.05), while an increase in the concentration of cysteine resulted in a significant improvement in the mitochondrial membrane potential, SOD activity, and ATP content (P < 0.05). As for lipid peroxidation, the extent of which in cysteine treated spermatozoa was less than the control, although the differences were not statistically significant (P > 0.05). In terms of fertilizing capacity, a greater hatching rate (91.7 ± 1.2%) was obtained in thawed sperm treated with 2 mM cysteine, compared to the control (84.3 ± 4.2%; P < 0.05). Overall, it is concluded that the addition of cysteine is helpful in maintaining the function of frozen-thawed brown-marbled grouper sperm, which can be recommended as an effective antioxidant to improve the semen cryopreservation efficiency. • Treatment with cysteine is effective in protecting brown-marbled grouper sperm from cryodamage. • The cytoprotective effect of cysteine exhibits a dose-dependent manner. • Fertilizing capacity of frozen-thawed brown-marbled grouper sperm is improved by supplementation of cysteine at 2 mM. [ABSTRACT FROM AUTHOR]

In this study, CuO, ZnS, and p-CuO/n-ZnS heterojunction composites were synthesized using a simple co-precipitation method for investigating their photocatalytic properties for dye degradation and fluorescence sensing of thiol-containing amino acids at room temperature. The synthesized CuO, ZnS, and p-CuO/n-ZnS heterostructures were analyzed using UV–vis, PL, XRD, and TEM. The results showed that the heterostructure exhibited enhanced absorption in the visible light range, and synthesized materials have spherical shape with an average particle size is 12 ± 5 nm. The photocatalytic activities of CuO, ZnS, and p-CuO/n-ZnS heterostructures were evaluated by the degradation of various dyes such as MB, MO, and MR under visible light irradiation. The p-CuO/n-ZnS heterostructure showed the best photocatalytic efficiency, with degradation rates of 98.3%, 94.2%, and 92.8% for MB, MO, and MR dyes, respectively, compared to CuO NPs (72.5%, 75.8%, and 73.5%) and ZnS NPs (76.2%, 79.1%, and 78.3%) within a 35 min of reaction. The enhanced photocatalytic efficiency of the p-CuO/n-ZnS heterostructure was attributed to its high charge separation efficiency, which remained stable for up to five cycles without any significant loss of activity. Furthermore, the p-CuO/n-ZnS heterostructure was used as a fluorescent sensor for detecting thiol-containing amino acids such as cysteine (Cys) and methionine (Met). The results showed that the heterostructure had good sensing performance for Cys and Met within the range of 1–100 μM, with a detection limit of 2.52 μM (Cys) and 5.14 μM (Met) based on the linear calibration range. Moreover, p-CuO/n-ZnS composite was used to detect Met in human serum and urine samples. [Display omitted] • Constructed CuO, ZnS, and p-CuO/n-ZnS composites using coprecipitation method. • Spherical shaped CuO, ZnS, and p-CuO/n-ZnS have monoclinic and zinc blend phase. • p-CuO/n-ZnS composite have excellent photodegradation of dyes under visible light. • p-CuO/n-ZnS composite have prominent stability and main active species are • O 2. • p-CuO/n-ZnS composite exhibited prominent sensing of amino acids (Cys and Met). [ABSTRACT FROM AUTHOR]

The prevalence of cystine urolithiasis in domestic ferrets (Mustela putorius furo) has increased dramatically in the past decade. Genetic mutations associated with a defect in the active resorption of cysteine (which is oxidized to cystine) and dibasic amino acids in the proximal renal tubules leading to cystinuria were described in several species; however, the etiology in ferrets is unknown. This study aimed to characterize cystinuria in pet ferrets. Urine samples were aseptically acquired from 27 domestic client-owned ferrets with known dietary and medical histories. The samples underwent a routine urinalysis and microscopic evaluation. Urine aliquots were deproteinized with 6% sulfosalicylic acid and analyzed for amino acid concentrations by ion exchange chromatography. Urine cystine concentrations ranged from 8 to 2486 nmol/mL (median 243; SD 540.02). Principal component analyses revealed correlations among urinary amino acid concentrations, which were explained mostly by three components. Analyses revealed significant correlations between cystine and citrulline, ornithine, and lysine concentrations. Linear regression revealed a negative correlation between age and urinary cystine concentrations and no significant correlation between cystinuria and diet or treats. This is the 1st description of urine amino acid analysis in a cohort of pet ferrets. The detected correlations between cystine, ornithine, and citrulline could indicate that the pathology responsible for cystine urolithiasis in ferrets is like in other species. Result may indicate young ferrets are at increased risk of cystine urolithiasis Further research is needed to evaluate the possible association between diet and cystinuria. [ABSTRACT FROM AUTHOR]

Fluoride contamination in water due to natural and human activities has become a significant problem worldwide due to the diseases caused by this pollutant, such as dental and skeletal fluorosis caused by constant exposure concentrations above 1 ppm. It is possible to design materials that efficiently absorb fluoride from water, one possibility being metal-organic frameworks (MOFs). The HKUST-1 is a copper-based MOF with a high surface area; however, this framework is unstable in an aqueous solution. In this work, we studied the functionalization of HKUST-1, using five amino acids (tyrosine, serine, histidine, proline, and cysteine) to stabilize the 3D structure in an aqueous medium and evaluate its fluoride adsorption capacity--the X-ray diffraction patterns of functionalized MOF with 5 and 20 wt.% amino acids show the same structure of HKUST-1 for all materials. The amount of amino acid present in each MOF was determined by the relative intensities of the diffraction peaks, and Fourier-transform infrared spectroscopy identified the characteristic bands of the N-H bond. The samples functionalized with 20 wt.% increased the hydrophobic nature, increasing thermal stability to 110°C (proline). The maximum fluoride adsorption was 0.44 mg·g-1 (40% for 5 wt.% cysteine-MOF) and 0.55 mg·g-1 (50% for 20 wt.% histidine-MOF). The MOF structure is stable after fluoride adsorption when functionalized with 5% of serine, proline, and cysteine and 20% of tyrosine, proline, histidine, and cysteine. [ABSTRACT FROM AUTHOR]

• 7 CPP genes were identified in the bean genome. • Orthologous relationships between the bean, G. max and Arabidopsis CPP genes indicated that these plants are evolutionarily linked. • Salt, drought and melatonin treatments caused differences in the expression levels of PvCPP genes. • Meltonin improves the negative effect of salt and drought stress. Cysteine-rich polycomb-like protein (CPP) transcription factors, which are prevalent in plants and animals but absent in prokaryotes and fungi, play a significant role in response to various abiotic stresses and the regulation of cell division and development of reproductive tissues in plants. Plants can not avoid environmental challenges because they are soil-bound organisms. Therefore, they activate physiological, pharmacological, and molecular stress defence mechanisms. Among the molecular defence systems is the repression or stimulation of the expression of specific genes or gene families by particular transcription factors. Identification of these gene families is of great importance. The CPP gene family has been identified in some plants but not in beans. This study aimed to identify and characterize the CPP genes in the P. vulgaris genome and investigate them using various bioinformatics tools on a genome-wide scale. Expression levels of melatonin and stress factors PvCPP genes were determined by qRT-PCR analysis. Seven CPP genes have been identified in the Phaseolus vulgaris genome. Except for the PvCPP7 gene, the other PvCPP genes contain two CXC domains. The CPP genes of Phaseolus vulgaris, Arabidopsis thaliana , and Glycine max were analyzed for their phylogenetic relationships and were separated into three distinct groups. In qRT-PCR analysis, 5 CPP genes were studied and their expression levels in stress and melatonin applications were determined and differences in expression levels of PvCPP genes were observed in all treatments. The information generated from this study will serve as a foundation for future functional genomics research by understanding the structure and function of CPP genes in bean genotypes. [ABSTRACT FROM AUTHOR]

In animals, adaptation to changes in cellular oxygen levels is coordinated largely by 2-oxoglutarate-dependent prolylhydroxylase domain (PHD) dioxygenase family members, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of nontranscription factor substrates; the regulators of G-protein signaling 4, 5. and 16 and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their coevolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2- sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO, and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia. Collectively, our comparative analysis provides further insight into ADO/N-degron-mediated oxygen sensing and its integration into established mechanisms of oxygen homeostasis. [ABSTRACT FROM AUTHOR]

Cells respond to amino acid depletion by activating stress responses. A recent study by Swanda et al. reveals that a decrease in lysosomal cystine triggers a novel stress response that transcriptionally activates ATF4 and protects cells from ferroptosis. A synthetic mRNA, CysRx, can prevent ATF4 activation and enhance antitumor effects. [ABSTRACT FROM AUTHOR]

NAD+ and glutathione precursors are currently used as metabolic modulators for improving the metabolic conditions associated with various human diseases, including non-alcoholic fatty liver disease, neurodegenerative diseases, mitochondrial myopathy, and age-induced diabetes. Here, we performed a one-day double blinded, placebo-controlled human clinical study to assess the safety and acute effects of six different Combined Metabolic Activators (CMAs) with 1 g of different NAD+ precursors based on global metabolomics analysis. Our integrative analysis showed that the NAD+ salvage pathway is the main source for boosting the NAD+ levels with the administration of CMAs without NAD+ precursors. We observed that incorporation of nicotinamide (Nam) in the CMAs can boost the NAD+ products, followed by niacin (NA), nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), but not flush free niacin (FFN). In addition, the NA administration led to a flushing reaction, accompanied by decreased phospholipids and increased bilirubin and bilirubin derivatives, which could be potentially risky. In conclusion, this study provided a plasma metabolomic landscape of different CMA formulations, and proposed that CMAs with Nam, NMN as well as NR can be administered for boosting NAD+ levels to improve altered metabolic conditions. [Display omitted] • The administration of serine, NAC and LCAT with NAD precursor is safe and well tolerated. • NAD salvage pathway is the main source of NAD when orally taking serine, NAC and LCAT. • Nam in the CMAs showed the best ability to boost plasma NAD+ products. • NA in the CMAs led to flushing reaction, decreased phospholipids and increased bilirubin. • Nam, NMN as well as NR are good candidates for boosting NAD level. [ABSTRACT FROM AUTHOR]

Growing evidence suggests that the depletion of plasma NAD+ and glutathione (GSH) may play an important role in the development of metabolic disorders. The administration of Combined Metabolic Activators (CMA), consisting of GSH and NAD+ precursors, has been explored as a promising therapeutic strategy to target multiple altered pathways associated with the pathogenesis of the diseases. Although studies have examined the therapeutic effect of CMA that contains N-acetyl- l -cysteine (NAC) as a metabolic activator, a system-wide comparison of the metabolic response to the administration of CMA with NAC and cysteine remains lacking. In this placebo-controlled study, we studied the acute effect of the CMA administration with different metabolic activators, including NAC or cysteine with/without nicotinamide or flush free niacin, and performed longitudinal untargeted-metabolomics profiling of plasma obtained from 70 well-characterized healthy volunteers. The time-series metabolomics data revealed the metabolic pathways affected after the administration of CMAs showed high similarity between CMA containing nicotinamide and NAC or cysteine as metabolic activators. Our analysis also showed that CMA with cysteine is well-tolerated and safe in healthy individuals throughout the study. Last, our study systematically provided insights into a complex and dynamics landscape involved in amino acid, lipid and nicotinamide metabolism, reflecting the metabolic responses to CMA administration containing different metabolic activators. [Display omitted] • The administration of different CMA formulas was safe and no toxic effect was seen in the healthy subjects participated in the study. • Similar plasma cysteine kinetics were observed between CMA formulas with NAC and cysteine. • Longitudinal untargeted-metabolomics profiling reveals the acute metabolic response is similar between CMA formulas with NAC and cysteine. [ABSTRACT FROM AUTHOR]

The antibody-linked oxi-state assay (ALISA) for quantifying target-specific cysteine oxidation can benefit specialist and non-specialist users. Specialists can benefit from time-efficient analysis and high-throughput target and/or sample n-plex capacities. The simple and accessible "off-the-shelf" nature of ALISA brings the benefits of oxidative damage assays to non-specialists studying redox-regulation. Until performance benchmarking establishes confidence in the "unseen" microplate results, ALISA is unlikely to be widely adopted. Here, we implemented pre-set pass/fail criteria to benchmark ALISA by robustly evaluating immunoassay performance in diverse biological contexts. ELISA-mode ALISA assays were accurate, reliable, and sensitive. For example, the average inter-assay CV for detecting 20%- and 40%-oxidised PRDX2 or GAPDH standards was 4.6% (range: 3.6–7.4%). ALISA displayed target-specificity. Immunodepleting the target decreased the signal by ∼75%. Single-antibody formatted ALISA failed to quantify the matrix-facing alpha subunit of the mitochondrial ATP synthase. However, RedoxiFluor quantified the alpha subunit displaying exceptional performance in the single-antibody format. ALISA discovered that (1) monocyte-to-macrophage differentiation amplified PRDX2-specific cysteine oxidation in THP-1 cells and (2) exercise increased GAPDH-specific cysteine oxidation in human erythrocytes. The "unseen" microplate data were "seen-to-be-believed" via orthogonal visually displayed immunoassays like the dimer method. Finally, we established target (n = 3) and sample (n = 100) n-plex capacities in ∼4 h with 50–70 min hands-on time. Our work showcases the potential of ALISA to advance our understanding of redox-regulation and oxidative stress. [Display omitted] • ALISA quantifies target-specific cysteine oxidation in a microplate. • ALISA is accurate, reliable, and sensitive. • ALISA generated new insights in diverse biological contexts. • The unseen microplate results were visually verified. • Specialists and non-specialists alike can benefit from ALISA. [ABSTRACT FROM AUTHOR]

Bioactivation of trichloroethylene (TCE) via glutathione conjugation is associated with several adverse effects in the kidney and other extrahepatic tissues. Of the three regioisomeric conjugates formed, S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione and S-(2,2-dichlorovinyl)-glutathione, only 1,2-trans-DCVG and its corresponding cysteine-conjugate, 1,2-trans-DCVC, have been subject to extensive mechanistic studies. In the present study, the metabolism and cellular effects of 1,2-cis-DCVG, the major regioisomer formed by rat liver fractions, and 1,2-cis-DCVC were investigated for the first time using RPTEC/TERT1-cells as in vitro renal model. In contrast to 1,2-trans-DCVG/C, the cis-regioisomers showed minimal effects on cell viability and mitochondrial respiration. Transcriptomics analysis showed that both 1,2-cis-DCVC and 1,2-trans-DCVC caused Nrf2-mediated antioxidant responses, with 3 µM as lowest effective concentration. An ATF4-mediated integrated stress response and p53-mediated responses were observed starting from 30 µM for 1,2-trans-DCVC and 125 µM for 1,2-cis-DCVC. Comparison of the metabolism of the DCVG regioisomers by LC/MS showed comparable rates of processing to their corresponding DCVC. No detectable N-acetylation was observed in RPTEC/TERT1 cells. Instead, N-glutamylation of DCVC to form N-γ-glutamyl-S-(dichlorovinyl)- L -cysteine was identified as a novel route of metabolism. The results suggest that 1,2-cis-DCVC may be of less toxicological concern for humans than 1,2-trans-DCVC, considering its lower intrinsic toxicity and lower rate of formation by human liver fractions. • First evaluation of effects of 1,2-cis-DCVC and 1,2-cis-DCVG using RPTEC/TERT1-cells. • 1,2-cis-DCVC shows lower cytotoxic effects compared to 1,2-trans-DCVC. • 1,2-cis-DCVC and 1,2-trans-DCVC showed similar Nrf2-stress responses. • 1,2-cis-DCVC was less potent to activate ATF4 and p53 controlled stress responses. • N-Glutamylation is identified as novel route of metabolism of DCVC. [ABSTRACT FROM AUTHOR]

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that results from mutations in NOTCH3. How mutations in NOTCH3 ultimately result in disease is not clear, although there is a predilection for mutations to alter the number of cysteines of the gene product, supporting a model in which alterations of conserved disulfide bonds of NOTCH3 drives the disease process. We have found that recombinant proteins with CADASIL NOTCH3 EGF domains 1 to 3 fused to the C terminus of Fc are distinguished from wildtype proteins by slowed mobility in nonreducing gels. We use this gel mobility shift assay to define the effects of mutations in the first three EGF-like domains of NOTCH3 in 167 unique recombinant protein constructs. This assay permits a readout on NOTCH3 protein mobility that indicates that (1) any loss of cysteine mutation in the first three EGF motifs results in structural abnormalities; (2) for loss of cysteine mutants, the mutant amino acid residue plays a minimal role; (3) the majority of changes that result in a new cysteine are poorly tolerated; (4) at residue 75, only cysteine, proline, and glycine induce structural shifts; (5) specific second mutations in conserved cysteines suppress the impact of loss of cysteine CADASIL mutations. These studies support the importance of NOTCH3 cysteines and disulfide bonds in maintaining normal protein structure. Double mutant analysis suggests that suppression of protein abnormalities can be achieved through modification of cysteine reactivity, a potential therapeutic strategy. [ABSTRACT FROM AUTHOR]

House dust mite (HDM) encloses an explosive cocktail of allergenic proteins sensitizing hundreds of millions of people worldwide. To date, the innate cellular and molecular mechanism(s) orchestrating the HDM-induced allergic inflammation remains partially deciphered. Understanding the kaleidoscope of HDM-induced innate immune responses is hampered by (1) the large complexity of the HDM allergome with very diverse functional bioreactivities, (2) the perpetual presence of microbial compounds (at least LPS, β-glucan, chitin) promoting as well pro-Th2 innate signaling pathways and (3) multiple cross-talks involving structural, neuronal and immune cells. The present review provides an update on the innate immune properties, identified so far, of multiple HDM allergen groups. Experimental evidence highlights the importance of HDM allergens displaying protease or lipid-binding activities on the initiation of the allergic responses. Specifically, group 1 HDM cysteine proteases are considered as the key initiators of the allergic response through their capacities to impair the epithelial barrier integrity, to stimulate the release of pro-Th2 danger-associated molecular patterns (DAMPs) in epithelial cells, to produce super-active forms of IL-33 alarmin and to mature thrombin leading to Toll-like receptor 4 (TLR4) activation. Remarkably, the recently evidenced primary sensing of cysteine protease allergens by nociceptive neurons confirms the critical role of this HDM allergen group in the early events leading to Th2 differentiation. • The functions of several HDM allergens promote pro-Th2 innate signaling pathways • HDM cysteine protease allergens stimulate the thrombin-PAR1/PAR4-TLR4 axis • HDM cysteine protease allergens are sensed by TRPV1 + sensory neurons • HDM group 2 allergens are lipid binding proteins sensed by TLR2 or TLR4 [ABSTRACT FROM AUTHOR]

The small molecule erastin inhibits the cystine-glutamate antiporter, system xc -, which leads to intracellular cysteine and glutathione depletion. This can cause ferroptosis, which is an oxidative cell death process characterized by uncontrolled lipid peroxidation. Erastin and other ferroptosis inducers have been shown to affect metabolism but the metabolic effects of these drugs have not been systematically studied. To this end, we investigated how erastin impacts global metabolism in cultured cells and compared this metabolic profile to that caused by the ferroptosis inducer RAS-selective lethal 3 or in vivo cysteine deprivation. Common among the metabolic profiles were alterations in nucleotide and central carbon metabolism. Supplementing nucleosides to cysteine-deprived cells rescued cell proliferation in certain contexts, showing that these alterations to nucleotide metabolism can affect cellular fitness. While inhibition of the glutathione peroxidase GPX4 caused a similar metabolic profile as cysteine deprivation, nucleoside treatment did not rescue cell viability or proliferation under RAS-selective lethal 3 treatment, suggesting that these metabolic changes have varying importance in different scenarios of ferroptosis. Together, our study shows how global metabolism is affected during ferroptosis and points to nucleotide metabolism as an important target of cysteine deprivation. [ABSTRACT FROM AUTHOR]

Reactive electrophilic species are ubiquitous in plant cells, where they contribute to specific redox-regulated signaling events. Redox signaling is known to modulate gene expression during diverse biological processes, including plant growth, development, and environmental stress responses. Emerging data demonstrates that transcription factors (TFs) are a main target of cysteine thiol-based oxidative post-translational modifications (OxiPTMs), which can alter their transcriptional activity and thereby convey redox information to the nucleus. Here, we review the significant progress that has been made in characterizing cysteine thiol-based OxiPTMs, their biochemical properties, and their functional effects on plant TFs. We discuss the underlying mechanism of redox regulation and its contribution to various physiological processes as well as still outstanding challenges in redox regulation of plant gene expression. Reactive electrophilic species, including reactive oxygen, nitrogen, and sulfur species (ROS/RNS/RSS), actively contribute to redox signal transduction in diverse signaling processes. Hydrogen peroxide (H 2 O 2), nitric oxide (NO), and hydrogen sulfide (H 2 S) are the most studied ROS/RNS/RSS, respectively. The biological function of H 2 O 2 /NO/H 2 S is largely governed through oxidative post-translational modifications (OxiPTMs) of proteins that impact protein function. An expanding list of transcription factors undergoes OxiPTMs and has been found to contribute to redox signaling–regulated transcription reprogramming in plant development and stress responses. OxiPTMs can alter the DNA binding ability, subcellular localization, protein stability, phase separation, and/or protein–protein interactions of transcription factors. [ABSTRACT FROM AUTHOR]

[Display omitted] • Curcumin improves systemic inflammation in subjects residing in an air-polluted area. • Curcumin mitigates acrolein-induced pneumonitis in a preclinical animal model. • Curcumin inhibits acrolein-induced alveolar epithelial apoptosis in a ROS-dependent manner. • Curcumin increases redox sensing of Nrf2 and thus sensitizes de novo GSH synthesis. • Curcumin-Keap1 cysteine conjugation enhances redox sensing of Nrf2. Epidemiological studies have reported an association between exposures to ambient air pollution and respiratory diseases, including chronic obstructive pulmonary disease (COPD). Pneumonitis is a critical driving factor of COPD and exposure to air pollutants (e.g., acrolein) is associated with increased incidence of pneumonitis. Currently available anti-inflammatory therapies provide little benefit against respiratory diseases. To this end, we investigated the preventive role of curcumin against air pollutant-associated pneumonitis and its underlying mechanism. A total of 40 subjects was recruited from Chengdu, China which is among the top three cities in terms of respiratory mortality related to air pollution. The participants were randomly provided either placebo or curcumin supplements for 2 weeks and blood samples were collected at the baseline and at the end of the intervention to monitor systemic markers. In our follow up mechanistic study, C57BL/6 mice (n = 40) were randomly allocated into 4 groups: Control group (saline + no acrolein), Curcumin only group (curcumin + no acrolein), Acrolein only group (saline + acrolein), and Acrolein + Curcumin group (curcumin + acrolein). Curcumin was orally administered at 100 mg/kg body weight once a day for 10 days, and then the mice were subjected to nasal instillation of acrolein (5 mg/kg body weight). Twelve hours after single acrolein exposure, all mice were euthanized. Curcumin supplementation, with no noticeable adverse responses, reduced circulating pro-inflammatory cytokines in association with clinical pneumonitis as positive predictive while improving those of anti-inflammatory cytokines. In the pre-clinical study, curcumin reduced pneumonitis manifestations by suppression of intrinsic and extrinsic apoptotic signaling, which is attributed to enhanced redox sensing of Nrf2 and thus sensitized synthesis and restoration of GSH, at least in part, through curcumin-Keap1 conjugation. Our study collectively suggests that curcumin could provide an effective preventive measure against air pollutant-enhanced pneumonitis and thus COPD. [ABSTRACT FROM AUTHOR]

CuO and CuO@Si core-shell nanoparticles were synthesized by hydrothermal method and analyzed their structural, chemical and morphological properties using XRD, FESEM, TEM, UV–Vis, FT-IR, and BET analysis. CuO and CuO@Si core-shell nanoparticles are appeared as spherical and homogeneous in size, with respective diameters of 17 and 80 nm in FE-SEM analysis. This developed nanomaterial was used to modify the electrode and as an efficient electrochemical strategic tool for the trace-level detection of l -cysteine. The cyclic voltammetry (CV), differential pulse voltammetry (DPV), and amperometry studies indicated that the CuO@Si core-shell nanoparticles show remarkable sensitivity for l -cysteine. The modified glassy carbon electrode shows linearity with the addition of increasing concentration (20–240 μL) of l -cysteine addition, which yields the detection limit of 0.015 μM. The CuO@SiO 2 core-shell nanoparticle is simple, rapidly synthesized, reliable, and the adopted synthetic method did not require extensive sample treatment. These nanomaterials have been utilized as potential anticancer agents against A549 & HEK 293 cell lines based on their MTT assay observations. The obtained antibacterial and anticancer properties have demonstrated that they are remarkably potent to inhibit bacterial growth, induce effective apoptosis in cancer cells relative to healthy cells, and also their environmental safety. [ABSTRACT FROM AUTHOR]

The tick-transmitted apicomplexan Theileria par va causes East Coast fever, a bovine disease of great economic and veterinary importance in Africa. Papain-like cysteine proteases play important roles in protozoan parasite host cell entry and egress, nutrition and host immune evasion. This study reports the identification and characterisation of a T. parva strain Muguga cathepsin L-like (C1A subfamily) cysteine protease (Thp CP). Molecular modelling confirmed the papain-like fold of Thp CP , hydrophobic character of the S2 substrate binding pocket and non-covalent interaction between the pro- and catalytic domains preceding low pH autoactivation. Thp CP was recombinantly expressed in a protease deficient E. coli (Rosetta (DE3)pLysS strain) expression host as a 46 kDa proenzyme. Following Ni-chelate affinity chromatography and acidification, the 27 kDa mature Thp CP was purified by cation-exchange chromatography. Purified Thp CP hydrolysed typical cathepsin L substrates N-α-benzyloxycarbonyl (Z)-Phe-Arg-7-amino-4-methyl-coumarin (AMC) (k cat / K m = 4.49 × 105 s−1M−1) and Z-Leu-Arg-AMC (k cat / K m = 4.20 × 105 s−1M−1), but showed no activity against the cathepsin B-selective substrate Z-Arg-Arg-AMC. Recombinant Thp CP was active over a broad pH range from pH 4.5 to 7.5, thereby showing potential activity in the acidic parasite food vacuole and close to neutral pH of the host lymphocyte cytoplasm. Recombinant Thp CP was inhibited by the cysteine protease inhibitors E64, iodoacetate, leupeptin, chymostatin, Z-Phe-Ala-diazomethylketone (DMK) and Z-Phe-Phe-DMK and hydrolysed bovine proteins: haemoglobin, immunoglobulin G, serum albumin and fibrinogen as well as goat IgG at pH 6 and 7. Functional expression and characterisation of Theileria cysteine proteases should enable high throughput screening of cysteine protease inhibitor libraries against these proteases. [Display omitted] • Cathepsin L-like cysteine protease from Theileria parva (Thp CP) characterised. • Molecular modelling showed papain-fold and hydrophobic S2 subsite. • Possible pro-domain—catalytic domain interactions precede activation at low pH. • Broad pH activity includes those of food vacuole and host lymphocyte cytoplasm. • Thp CP hydrolyses host haemoglobin, immunoglobulin, fibrinogen and serum albumin. [ABSTRACT FROM AUTHOR]

l -Methionine biosynthesis is through multilevel regulated and multibranched biosynthetic pathway (MRMBP). Because of the complex regulatory mechanism and the imbalanced metabolic flux between branched pathways, microbial production of l -methionine has not been commercialized. In this study, local metabolic response in MRMBP of l -methionine was investigated and various crucial genes in branched pathways were determined. In l -serine pathway, the crucial gene was serABC. In O -succinyl homoserine (OSH) pathway, which was the C4 backbone of l -methionine, metB and metL controlled the metabolic flux jointly. In l -cysteine pathway, the crucial gene cysE fbr could disturb the flux distribution of local network in l -methionine biosynthesis. However, no crucial gene for l -methionine production in 5-methyl tetrahydrofolate (CH 3 -THF) pathway was found. The relation between these pathways was also researched. l -Serine pathway, as the upstream pathway of l -cysteine and CH 3 -THF, played a crucial role in l -methionine biosynthesis. l -Cysteine pathway showed the strongest controlling force of the metabolic flux, and OSH pathway was second to l -cysteine pathway. In contrast, CH 3 -THF pathway was the weakest, which was probably the mainly limited steps at present and had great potential in further research. In addition, constructed W3110 IJAHFEBC/pA∗HAmL was able to produce 2.62 g/L l -methionine in flask. This study is instructive for l -methionine biosynthesis and provides a new research method of biosynthesizing other metabolic products in MRMBPs. [Display omitted] • Multilevel regulated and multibranched biosynthetic pathways (MRMBPs) for l -methionine biosynthesis were analyzed. • Various crucial genes in MRMBPs for l -methionine biosynthesis were determined. • The balance between MRMBPs for l -methionine biosynthesis was clarified. [ABSTRACT FROM AUTHOR]

Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-dependent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is protected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "β-latch" structural element that includes the α6 helix, a glycine-rich loop, a β-hairpin, and a cis-proline residue. To identify a functional role for the β-latch, we used sitedirected mutagenesis to obtain the N99D and N99A SufS variants. N99 is a conserved residue that connects the α6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a distorted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a highaffinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demonstrate that the β-latch does not affect the chemistry of persulfide formation but does protect it from undesired reductants. The data also indicate the β-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer. [ABSTRACT FROM AUTHOR]

In this study, a novel bioadhesive material, a conjugate of chondroitin sulfate and L-cysteine (CS-Cys), was synthesized and modified on the surface of the cationic nanostructured lipid carriers loaded dexamethasone to prepare a novel nano-lipid ocular delivery system (Dex-CS-Cys-cNLC). Through the permeation and retention studies of isolated cornea, it was demonstrated that Dex-CS-Cys-cNLC has better corneal permeation and retention ability and can better overcome the barrier effect of the ocular surface. In addition, the fluorescent probe (RhB) was used to replace the drug, and fluorescence imaging was used to investigate the ocular surface retention ability of the formulation, and the results showed that CS-Cys-cNLC has stronger retention ability and can effectively prolong the time of drug action in the ocular surface. Dex-CS-Cys-cNLC was not irritating to rabbit eye tissues and was a safe delivery system. The results of rabbit dry eye pharmacodynamic experiments also showed that Dex-CS-Cys-cNLC could effectively alleviate dry eye symptoms in rabbits, effectively repair corneal damage, and improve the stability of tear film. All these experimental results suggest that Dex-CS-Cys-cNLC is a promising drug delivery carrier for the treatment of anterior segment of the eye disease. [ABSTRACT FROM AUTHOR]

Deep learning algorithms such as AlphaFold2 predict three-dimensional protein structure with high confidence. The recent release of more than 200 million structural models provides an unprecedented resource for functional protein annotation. Here, we used AlphaFold2 predicted structures of fifteen plant proteomes to functionally and evolutionary analyze cysteine residues in the plant kingdom. In addition to identification of metal ligands coordinated by cysteine residues, we systematically analyzed cysteine disulfides present in these structural predictions. Our analysis demonstrates most of these predicted disulfides are trustworthy due their high agreement (∼96%) with those present in X-ray and NMR protein structures, their characteristic disulfide stereochemistry, the biased subcellular distribution of their proteins and a higher degree of oxidation of their respective cysteines as measured by proteomics. Adopting an evolutionary perspective, zinc binding sites are increasingly present at the expense of iron-sulfur clusters in plants. Interestingly, disulfide formation is increased in secreted proteins of land plants, likely promoting sequence evolution to adapt to changing environments encountered by plants. In summary, Alphafold2 predicted structural models are a rich source of information for studying the role of cysteines residues in proteins of interest and for protein redox biology in general. [Display omitted] • AlphaFold2 predictions enable proteome-wide structural annotation of cysteines forming disulfides or binding metal ligands. • Disulfide formation is highly compartmentalized and in line with the subcellular redox environment. • Zinc binding sites are increasingly present throughout plant evolution, at the expense of iron-sulfur clusters. • Secreted proteins in land plants are richer in disulfides, likely facilitating adaptations to terrestrial habitats. • Protein structure predictions can hint towards intermolecular disulfides and cysteine-bound cofactors such as heme. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS), released as byproducts of mitochondrial metabolism or as products of NADPH oxidases and other processes, can directly oxidize the active-site cysteine (Cys) residue of protein tyrosine phosphatases (PTPs) in a mammalian cell. Robust degradation of irreversibly oxidized PTPs is essential for preventing accumulation of these permanently inactive enzymes. However, the mechanism underlying the degradation of these proteins was unknown. In this study, we found that the active-site Cys215 of endogenous PTP1B is sulfonated in H9c2 cardiomyocytes under physiological conditions. The sulfonation of Cys215 led PTP1B to exhibit a conformational change, and drive the subsequent ubiquitination and degradation of this protein. We then discovered that Cullin1, an E3 ligase, interacts with the Cys215-sulfonated PTP1B. The functional impairment of Cullin1 prevented PTP1B from oxidation-dependent ubiquitination and degradation in H9c2 cells. Moreover, delivery of the terminally oxidized PTP1B resulted in proteotoxicity-caused injury in the affected cells. In conclusion, we elucidate how sulfonation of the active-site Cys215 can direct turnover of endogenous PTP1B through the engagement of ubiquitin-proteasome system. These data highlight a novel mechanism that maintains PTP homeostasis in cardiomyocytes with constitutive ROS production. [Display omitted] • Active-site Cys215-SO3 PTP1B is degraded by proteasome-mediated process. • Cys215-SO3 PTP1B interacts with Cullin1 E3 ligase for proteolysis. • Intracellular accumulation of Cys215-SO3 PTP1B leads to cytotoxicity. [ABSTRACT FROM AUTHOR]

• NaCl stress-induced restructuring of root architecture coincides with enhanced H 2 S accumulation. • Melatonin modulates H 2 S homeostasis in sunflower cotyledons and roots. • Exogenous Ca2+exerted differential effects on Mel-H 2 S signaling in control and NaCl stressed seedlings. • Activity of extracellular Ca2+influx channel is crucial for melatonin-induced endogenous H 2 S accumulation. Although Ca2+ has been known to be a modulator of abiotic stress signaling in plants, not much information is available to decipher the interactive roles of calcium and melatonin in mediating hydrogen sulphide homeostasis under NaCl stress. Present work provides evidence on the roles of exogenous calcium (Ca2+) supplementation and endogenous Ca2+ channel activity (verapamil-sensitive) in modulating melatonin-mediated effects on seedling growth and H 2 S homeostasis in NaCl-stressed sunflower seedlings. In this context, exogenous Ca2+ and verapamil treatments exert differential effects in cotyledons and roots of seedlings raised in the absence and presence of melatonin. On comparing the effects of exogenous Ca2+ supplementation and verapamil-sensitive Ca2+ channel activity, it was observed that exogenous Ca2+exerted differential effects on Mel-H 2 S signaling in control and NaCl stressed seedlings. Verapamil-sensitive channels seem to be crucial for mediating the effects of exogenous melatonin in sunflower seedlings. Present evidence also indicate that in the absence of exogenous Ca2+, endogenous Ca2+ channel activity and cytosolic Ca2+ milieu are essential for mediating melatonin-H 2 S signaling in sunflower seedlings. However, it is imperative to undertake future investigations on the probable interaction of melatonin, Ca2+-CaM and other Ca2+-binding proteins as possible regulators of H 2 S metabolism in plants [ABSTRACT FROM AUTHOR]

Quercetin (QCT) has antioxidant, anti-inflammatory, anti-tumor and other important pharmacological activities, but the poor water solubility limits its application. In this work, the amphiphilic dextran (ADEX) was prepared by grafting L-cysteine and octadecylamine onto carboxymethyl dextran with the grafting rate of 21.29 % and 19.35 %. Then, the QCT-loaded nanomicelles (QNMs) were prepared by using ADEX as wall material and the QCT as core material via ultrasonic self-assembly method. The particle size and zeta potential of QNMs were 372 nm and 31.4 mV. Under simulated gastric and simulated intestinal fluids, the cumulative release QNMs were 37.54 % and 52.13 % within 180 min, and the QNMs showed better stability in simulated gastric fluid. The QNMs showed significantly better PTIO , OH and O 2 − scavenging activities than QCT. In addition, QNMs could effectively down-regulate the expression of pro-inflammatory cytokines and promoted the expression of anti-inflammatory cytokine. The cellular uptake results proved that the QNMs were more easily absorbed by cells than free QCT, indicating that the nano-encapsulation procedure effectively improved the uptake efficiency of QCT by cells. • The amphiphilic dextran was prepared by one-pot method. • Quercetin nanomicelles (QNMs) was obtained by ultrasonic self-assembly method. • QNMs showed better stability in simulated gastric conditions than intestinal fluids. • QNMs showed higher antioxidant, anti-inflammatory and cellular uptake efficiency than free QCT. [ABSTRACT FROM AUTHOR]

[Display omitted] Chronic hepatic diseases often compromise liver function and are directly responsible for up to two million yearly deaths world-wide. There are yet no treatment options to solve this global medical need. Experimental drugs elafibranor (Ela) and obeticholic acid (OA) appeared promising in numerous earlier studies, but they recently struggled to show significant benefits in patients. Little is known on the drugs' impact on hepatic stellate cells (HSCs), key players in liver fibrogenesis. We recently reported a beneficial effect of polyenylphosphatidylcholines (PPCs)-rich formulations in reverting fibrogenic features of HSCs, including differences in their extracellular vesicles (EVs). Here, we newly formulated Ela and OA in PPC liposomes and evaluated their performance on the LX-2 (human HSC) cell line through our rigorous methods of EV-analysis, now expanded to include lipidomics. We show that direct treatments with Ela and OA increase EV-associated secreted protein acidic and cysteine rich (SPARC), a matricellular protein overexpressed in fibrogenesis. However, our results suggest that this potentially damaging drugs' action to HSCs could be mitigated when delivering them with lipid-based formulations, most notably with a PPC-rich phospholipid inducing specific changes in the cellular and EV phospholipid composition. Thus, EV analysis substantially deepens evaluations of drug performances and delivery strategies. [ABSTRACT FROM AUTHOR]

Compared to subcutaneous injections, oral administration of insulin would be a preferred route of drug administration for diabetic patients. For oral delivery, both liposomes and alginate hydrogels face many challenges, including early burst release of the encapsulated drug and poor intestinal drug absorption. Also, adhesion to the intestinal mucosa remains weak, which all result in a low bioavailability of the payload. This study reports on an alginate hydrogel loaded with liposomes for oral insulin administration. Liposomes (Lip) loaded with arginine-insulin complexes (AINS) were incorporated into a hydrogel prepared from cysteine modified alginate (Cys-Alg) to form liposome-in-alginate hydrogels (AINS-Lip-Gel). An ex vivo study proves that intestinal permeation of AINS and AINS-Lip is approximately 2.0 and 6.0-fold, respectively, higher than that of free insulin. The hydrogel retarded early release of insulin (∼30%) from the liposomes and enhanced the intestinal mucosal retention. In vivo experiments revealed that the AINS-Lip-Gel released insulin in a controlled manner and possessed strong hypoglycemic effects. We conclude that liposome-in-alginate hydrogels loaded with AINS represent an attractive strategy for the oral delivery of insulin. [Display omitted] • A novel liposome-in-hydrogel complex system was developed for oral delivery of insulin. • The modification of cysteine promotes mucosal retention of alginate. • Complex system only releases ∼10% insulin at pH 1.2. • Arginine modification and liposome encapsulation promotes intestinal absorption of insulin. [ABSTRACT FROM AUTHOR]

Hsp70s are multifunctional proteins and serve as the central hub of the protein quality control network. Hsp70s are also related to a number of diseases and have been established as drug targets. Human HspA1A (hHsp70) and HspA8 (hHsc70) are the major cytosolic Hsp70s, and they have both overlapping and distinct functions. hHsp70 contains five cysteine residues, and hHsc70 contains four cysteine residues. Previous studies have shown these cysteine residues can undergo different cysteine modifications such as oxidation or reaction with electrophiles to regulate their function, and hHsp70 and hHsc70 have different cysteine reactivity. To address the mechanism of the differences in cysteine reactivity between hHsp70 and hHsc70, we studied the factors that determine this reactivity by Ellman assay for the quantification of accessible free thiols and NMR analysis for the assessment of structural dynamics. We found the lower cysteine reactivity of hHsc70 is probably due to its lower structural dynamics and the stronger inhibition effect of interaction between the α-helical lid subdomain of the substrate-binding domain (SBDα) and the β-sheet substrate-binding subdomain (SBDβ) on cysteine reactivity of hHsc70. We determined that Gly557 in hHsp70 contributes significantly to the higher structural dynamics and cysteine reactivity of hHsp70 SBDα. Exploring the cysteine reactivity of hHsp70 and hHsc70 facilitates an understanding of the effects of redox reactions and electrophiles on their chaperone activity and regulation mechanisms, and how these differences allow them to undertake distinct cellular roles. [ABSTRACT FROM AUTHOR]

Designing smart electrodes is the key to the efficient water splitting for the production of large scale hydrogen as a clean energy source. In this study, we prepare an organic chiral molecule modified Au–Ni bilayer thin film electrode to examine the chiral induced spin selectivity (CISS) effect on water splitting. Electrodes with bilayer configuration consisting of thin Ni layer (100 nm) with an Au over layer (10 nm) are prepared on glass substrates by combined sputtering, thermal evaporation techniques. Subsequently, self-assembled monolayer of chiral L-Cysteine molecule is immobilized on the as-prepared Ni/Au surface by chemisorption method. The electrocatalytic behaviour of as-modified chiral electrodes (Ni/Au/Cys) has been investigated in 0.1 M KOH solution. Our results show that for achieving the current density of 5 mAcm−2 the reaction over potential decreases by 390 mV while 5-fold increase in the current density value is achieved at a fixed over potential with chiral Ni/Au/Cys thin film compared to the achiral (bare) bilayer Ni/Au thin film during oxygen evolution reaction (OER). The dramatic reduction of over potential for OER has been attributed to the spin specific reaction occurring at the chiral Ni/Au/Cys electrodes during water splitting. On the other hand, we observe that there is a decrease of 260 mV over potential with more than 11-fold increase in the absolute current density value (∼153 mAcm−2 at −0.6 V) for Ni/Au/Cys thin film than bare Ni/Au thin film in hydrogen evolution reaction (HER). The excellent bifunctional catalytic property of Ni/Au/Cys has been attributed to the synergistic effect of chirality and bilayer configuration present in the primary structure of cysteine molecule and Ni/Au thin films respectively. • L-Cysteine coated Au–Ni bilayer thin film device as a bifunctional catalyst. • CISS effect induced enhanced catalytic activities for OER and HER. • 390 mV decrease in over potential with 5-fold increase in current density for OER. • 260 mV decrease in over potential with 11-fold increase in current density for HER. • Efficient chiral molecule based device for water splitting applications. [ABSTRACT FROM AUTHOR]

Succination is the spontaneous reaction between the respiratory intermediate fumarate and cellular thiols that forms stable S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC). 2SC is a biomarker for conditions associated with elevated fumarate levels, including diabetes, obesity, and certain cancers, and succination likely contributes to disease progression. Bacillus subtilis has a yxe operon-encoded breakdown pathway for 2SC that involves three distinct enzymatic conversions. The first step is N-acetylation of 2SC by YxeL to form N-acetyl-2SC (2SNAC). YxeK catalyzes the oxygenation of 2SNAC, resulting in its breakdown to oxalo- acetate and N-acetylcysteine, which is deacetylated by YxeP to give cysteine. The monooxygenase YxeK is key to the pathway but is rare, with close homologs occurring infrequently in prokaryote and fungal genomes. The existence of additional 2SC breakdown pathways was not known prior to this study. Here, we used comparative genomics to identify a S-(2-succino) lyase (2SL) that replaces yxeK in some yxe gene clusters. 2SL genes from Enterococcus italicus and Dickeya dadantii complement B. subtilis yxeK mutants. We also determined that recombinant 2SL enzymes efficiently break down 2SNAC into fumarate and N-acetylcysteine, can perform the reverse reaction, and have minor activity against 2SC and other small molecule thiols. The strong preferences both YxeK and 2SL enzymes have for 2SNAC indicate that 2SC acetylation is a conserved breakdown step. The identification of a second naturally occurring 2SC breakdown pathway underscores the importance of 2SC catabolism and defines a general strategy for 2SC breakdown involving acetylation, breakdown, and deacetylation. [ABSTRACT FROM AUTHOR]