HDAC6 inhibitors sensitize non-mesenchymal triple-negative breast cancer cells to cysteine deprivation.

Triple-negative breast cancer (TNBC) is a highly malignant type of breast cancer and lacks effective therapy. Targeting cysteine-dependence is an emerging strategy to treat the mesenchymal TNBC. However, many TNBC cells are non-mesenchymal and unresponsive to cysteine deprivation. To overcome such resistance, three selective HDAC6 inhibitors (Tubacin, CAY10603, and Tubastatin A), identified by epigenetic compound library screening, can synergize with cysteine deprivation to induce cell death in the non-mesenchymal TNBC. Despite the efficacy of HDAC6 inhibitor, knockout of HDAC6 did not mimic the synthetic lethality induced by its inhibitors, indicating that HDAC6 is not the actual target of HDAC6 inhibitor in this context. Instead, transcriptomic profiling showed that tubacin triggers an extensive gene transcriptional program in combination with erastin, a cysteine transport blocker. Notably, the zinc-related gene response along with an increase of labile zinc was induced in cells by the combination treatment. The disturbance of zinc homeostasis was driven by PKCγ activation, which revealed that the PKCγ signaling pathway is required for HDAC6 inhibitor-mediated synthetic lethality. Overall, our study identifies a novel function of HDAC6 inhibitors that function as potent sensitizers of cysteine deprivation and are capable of abolishing cysteine-independence in non-mesenchymal TNBC.

Roles of cysteinyl leukotrienes and their receptors in immune cell-related functions.

The cysteinyl leukotrienes (cys-LTs), leukotriene C4, (LTC4), LTD4, and LTE4, are lipid mediators of inflammation. LTC4 is the only intracellularly synthesized cys-LT through the 5-lipoxygenase and LTC4 synthase pathway and after transport is metabolized to LTD4 and LTE4 by specific extracellular peptidases. Each cys-LT has a preferred functional receptor in vivo; LTD4 to the type 1 cys-LT receptor (CysLT1R), LTC4 to CysLT2R, and LTE4 to CysLT3R (OXGR1 or GPR99). Recent studies in mouse models revealed that there are multiple regulatory mechanisms for these receptor functions and each receptor plays a distinct role as observed in different mouse models of inflammation and immune responses. This review focuses on the integrated host responses to the cys-LT/CysLTR pathway composed of sequential ligands with preferred receptors as seen from mouse models. It also discusses potential therapeutic targets for LTC4 synthase, CysLT2R, and CysLT3R.

Functional analysis of YY1 zinc fingers through cysteine mutagenesis.

As a transcription factor, Yin Yang 1 (YY1) either activates or represses gene expression depending on its recruited cofactors. The YY1 C-terminal consists of four zinc fingers (ZF) that are responsible for its DNA binding. However, the contribution of each YY1 ZF to its functions have not been fully elucidated. In this study, we used alanines to replace YY1 cysteines that are crucial to ZFs in binding to DNA. We characterized these YY1 mutants for their DNA binding, transcriptional activity, and functional role in maintaining MDA-MB-231 cell proliferation. We demonstrated that ZFs 2 and 3 are essential to the general biological activity of YY1. ZF 1 showed relatively low importance, while ZF 4 is virtually dispensable for YY1 function.

Cysteine: A multifaceted amino acid involved in signaling, plant resistance and antifungal development.

Early effects induced by cysteine were monitored using the model of Mimosa pudica pulvinar cells. Rapid dose-dependent membrane depolarization (within seconds) and modification of proton secretion (within minutes) were triggered at cysteine concentrations higher than 0.1 mM. These effects did not result from a modification of the plasma membrane H+-ATPase activity nor from a protonophore effect as shown by assays on plasma membrane vesicles isolated from pulvinar tissues. In a 0.5-10 mM range, cysteine inhibited the ion-driven turgor-mediated seismonastic reaction of Mimosa pudica primary pulvini and the dark-induced movement of Cassia fasciculata leaflets. At concentrations higher than 1 mM, it induced a long-lasting leaflet necrosis dependent on the concentration and treatment duration. Electron microscopy showed that cysteine induced important damage in the nucleus, mitochondria, endoplasmic reticulum and Golgi of the M. pudica motor cell. Cysteine inhibited in a concentration-dependent manner, from 0.5 to 20 mM, both the mycelial growth and the spore germination of the fungal pathogens Phaeomoniella chlamydospora and Phaeoacremonium minimum implicated in esca disease of grapevines. Using [35S] cysteine, we showed that the amino acid was absorbed following leaf spraying, translocated from leaves to other parts of grapevine cuttings and accumulated within trunks and roots. Therefore, cysteine showed relevant properties to be a candidate able to control fungal diseases either by acting as an early signal directing plant host reaction or/and by acting directly on fungal development.

Using in vivo oxidation status of one- and two-component redox relays to determine H2O2 levels linked to signaling and toxicity.

BACKGROUND: Hydrogen peroxide (H2O2) is generated as a by-product of metabolic reactions during oxygen use by aerobic organisms, and can be toxic or participate in signaling processes. Cells, therefore, need to be able to sense and respond to H2O2 in an appropriate manner. This is often accomplished through thiol switches: Cysteine residues in proteins that can act as sensors, and which are both scarce and finely tuned. Bacteria and eukaryotes use different types of such sensors-either a one-component (OxyR) or two-component (Pap1-Tpx1) redox relay, respectively. However, the biological significance of these two different signaling modes is not fully understood, and the concentrations and peroxides driving those types of redox cascades have not been determined, nor the intracellular H2O2 levels linked to toxicity. Here we elucidate the characteristics, rates, and dynamic ranges of both systems. RESULTS: By comparing the activation of both systems in fission yeast, and applying mathematical equations to the experimental data, we estimate the toxic threshold of intracellular H2O2 able to halt aerobic growth, and the temporal gradients of extracellular to intracellular peroxides. By calculating both the oxidation rates of OxyR and Tpx1 by peroxides, and their reduction rates by the cellular redoxin systems, we propose that, while Tpx1 is a sensor and an efficient H2O2 scavenger because it displays fast oxidation and reduction rates, OxyR is strictly a H2O2 sensor, since its reduction kinetics are significantly slower than its oxidation by peroxides, and therefore, it remains oxidized long enough to execute its transcriptional role. We also show that these two paradigmatic H2O2-sensing models are biologically similar at pre-toxic peroxide levels, but display strikingly different activation behaviors at toxic doses. CONCLUSIONS: Both Tpx1 and OxyR contain thiol switches, with very high reactivity towards peroxides. Nevertheless, the fast reduction of Tpx1 defines it as a scavenger, and this efficient recycling dramatically changes the Tpx1-Pap1 response to H2O2 and connects H2O2 sensing to the redox state of the cell. In contrast, OxyR is a true H2O2 sensor but not a scavenger, being partially insulated from the cellular electron donor capacity.

The interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in mouse corpus cavernosum.

The aim of this study was to investigate the possible interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H2S substrate; 10-6-10-3 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10-6-10-3 M) and acetylcholine (10-9-10-4 M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 × 10-5 M), selective M1 mAChR antagonist pirenzepine (5 × 10-5 M) and selective M3 mAChR antagonist 4-DAMP (10-7 M) but not by selective M2 mAChR antagonist AF-DX 116 (10-6 M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10-2 M) and cystathionine-ß-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10-3 M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M1 and M3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded.

Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes plasma cholesteryl ester formation and is defective in familial lecithin:cholesterol acyltransferase deficiency (FLD), an autosomal recessive disorder characterized by low high-density lipoprotein, anemia, and renal disease. This study aimed to investigate the mechanism by which compound A [3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile], a small heterocyclic amine, activates LCAT. The effect of compound A on LCAT was tested in human plasma and with recombinant LCAT. Mass spectrometry and nuclear magnetic resonance were used to determine compound A adduct formation with LCAT. Molecular modeling was performed to gain insight into the effects of compound A on LCAT structure and activity. Compound A increased LCAT activity in a subset (three of nine) of LCAT mutations to levels comparable to FLD heterozygotes. The site-directed mutation LCAT-Cys31Gly prevented activation by compound A. Substitution of Cys31 with charged residues (Glu, Arg, and Lys) decreased LCAT activity, whereas bulky hydrophobic groups (Trp, Leu, Phe, and Met) increased activity up to 3-fold (P < 0.005). Mass spectrometry of a tryptic digestion of LCAT incubated with compound A revealed a +103.017 m/z adduct on Cys31, consistent with the addition of a single hydrophobic cyanopyrazine ring. Molecular modeling identified potential interactions of compound A near Cys31 and structural changes correlating with enhanced activity. Functional groups important for LCAT activation by compound A were identified by testing compound A derivatives. Finally, sulfhydryl-reactive ß-lactams were developed as a new class of LCAT activators. In conclusion, compound A activates LCAT, including some FLD mutations, by forming a hydrophobic adduct with Cys31, thus providing a mechanistic rationale for the design of future LCAT activators.

L-Cysteine Metabolism and Fermentation in Microorganisms.

L-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are industrially produced by microbial fermentation, L-cysteine has been mainly produced by protein hydrolysis. Due to environmental and safety problems, synthetic or biotechnological products have been preferred in the market. Here, we reviewed L-cysteine metabolism, including biosynthesis, degradation, and transport, and biotechnological production (including both enzymatic and fermentation processes) of L-cysteine. The metabolic regulation of L-cysteine including novel sulfur metabolic pathways found in microorganisms is also discussed. Recent advancement in biochemical studies, genome sequencing, structural biology, and metabolome analysis has enabled us to use various approaches to achieve direct fermentation of L-cysteine from glucose. For example, worldwide companies began to supply L-cysteine and its derivatives produced by bacterial fermentation. These companies successfully optimized the original metabolism of their private strains. Basically, a combination of three factors should be required for improving L-cysteine fermentation: that is, (1) enhancing biosynthesis: overexpression of the altered cysE gene encoding feedback inhibition-insensitive L-serine O-acetyltransferase (SAT), (2) weakening degradation: knockout of the genes encoding L-cysteine desulfhydrases, and (3) exploiting export system: overexpression of the gene involved in L-cysteine transport. Moreover, we found that "thiosulfate" is much more effective sulfur source than commonly used "sulfate" for L-cysteine production in Escherichia coli, because thiosulfate is advantageous for saving consumption of NADPH and relating energy molecules.

Novel Technologies for Optimal Strain Breeding.

The implementation of a knowledge-based bioeconomy requires the rapid development of highly efficient microbial production strains that are able to convert renewable carbon sources to value-added products, such as bulk and fine chemicals, pharmaceuticals, or proteins at industrial scale. Starting from classical strain breeding by random mutagenesis and screening in the 1950s via rational design by metabolic engineering initiated in the 1970s, a range of powerful new technologies have been developed in the past two decades that can revolutionize future strain engineering. In particular, next-generation sequencing technologies combined with new methods of genome engineering and high-throughput screening based on genetically encoded biosensors have allowed for new concepts. In this chapter, selected new technologies relevant for breeding microbial production strains with a special emphasis on amino acid producers will be summarized.

Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type: Distinct roles of cysteine residues in redox regulation and NADPH inhibition.

A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants.

Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin-like proteins of Archaea.

Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding, and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed that ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of autoregulation. Cysteine residues, distinct from the canonical E1 'active site' cysteine, were found important in UbaA function supporting a model that this noncanonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.

Role of Cysteinyl Leukotrienes in Allergic Rhinitis.

Cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic rhinitis. Pharmacological studies using CysLTs indicate that two classes of receptor exist: CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R). The CysLT1R is a high-affinity leukotriene D4 receptor with lower affinity for leukotriene C4 that is sensitive to the CysLT1R antagonist currently used to treat asthma and allergic rhinitis. Our previous immunohistochemical and autoradiographic studies have demonstrated the presence of anti-CysLT1R antibodies labeled in eosinophils, mast cells, macrophages, neutrophils and vascular endothelial cells in human nasal mucosa. Furthermore, we have revealed that the novel radioactive CysLT1R antagonist [3H]-pranlukast bound specifically to CysLT1R in human inferior turbinates and its binding sites were localized to vascular endothelium and the interstitial cells. These data suggest that the major targets of CysLT1R antagonists in allergic rhinitis are the vascular bed and infiltrated leukocytes such as mast cells, eosinophils and macrophages. Clinical trials have demonstrated that CysLT1R antagonists are as effective as antihistamines for the treatment of allergic rhinitis; however, they are less effective than intranasal steroids. The use of CysLT1R antagonists in combination with antihistamines has generally resulted in greater efficacy than when these agents were used alone.

Reactions of electrophiles with nucleophilic thiolate sites: relevance to pathophysiological mechanisms and remediation.

Electrophiles are electron-deficient species that form covalent bonds with electron-rich nucleophiles. In biological systems, reversible electrophile-nucleophile interactions mediate basal cytophysiological functions (e.g. enzyme regulation through S-nitrosylation), whereas irreversible electrophilic adduction of cellular macromolecules is involved in pathogenic processes that underlie many disease and injury states. The nucleophiles most often targeted by electrophiles are side chains on protein amino acids (e.g. Cys, His, and Lys) and aromatic nitrogen sites on DNA bases (e.g. guanine N7). The sulfhydryl thiol (RSH) side chain of cysteine residues is a weak nucleophile that can be ionized in specific conditions to a more reactive nucleophilic thiolate (RS(-)). This review will focus on electrophile interactions with cysteine thiolates and the pathophysiological consequences that result from irreversible electrophile modification of this anionic sulfur. According to the Hard and Soft, Acids and Bases (HSAB) theory of Pearson, electrophiles and nucleophiles can be classified as either soft or hard depending on their relative polarizability. HSAB theory suggests that electrophiles will preferentially and more rapidly form covalent adducts with nucleophiles of comparable softness or hardness. Application of HSAB principles, in conjunction with in vitro and proteomic studies, have indicated that soft electrophiles of broad chemical classes selectively form covalent Michael-type adducts with soft, highly reactive cysteine thiolate nucleophiles. Therefore, these electrophiles exhibit a common mechanism of cytotoxicity. As we will discuss, this level of detailed mechanistic understanding is a necessary prerequisite for the rational development of effective prevention and treatment strategies for electrophile-based pathogenic states.

Local and systemic vasodilatory effects of low molecular weight S-nitrosothiols.

S-nitrosothiols (SNOs) such as S-nitroso-L-cysteine (L-cysNO) are endogenous compounds with potent vasodilatory activity. During circulation in the blood, the NO moiety can be exchanged among various thiol-containing compounds by S-transnitrosylation, resulting in SNOs with differing capacities to enter the cell (membrane permeability). To determine whether the vasodilating potency of SNOs is dependent upon membrane permeability, membrane-permeable L-cysNO and impermeable S-nitroso-D-cysteine (D-cysNO) and S-nitroso-glutathione (GSNO) were infused into one femoral artery of anesthetized adult sheep while measuring bilateral femoral and systemic vascular conductances. L-cysNO induced vasodilation in the infused hind limb, whereas D-cysNO and GSNO did not. L-cysNO also increased intracellular NO in isolated arterial smooth muscle cells, whereas GSNO did not. The infused SNOs remained predominantly in a low molecular weight form during first-passage through the hind limb vasculature, but were converted into high molecular weight SNOs upon systemic recirculation. At systemic concentrations of ~0.6 µmol/L, all three SNOs reduced mean arterial blood pressure by ~50%, with pronounced vasodilation in the mesenteric bed. Pharmacokinetics of L-cysNO and GSNO were measured in vitro and in vivo and correlated with their hemodynamic effects, membrane permeability, and S-transnitrosylation. These results suggest local vasodilation by SNOs in the hind limb requires membrane permeation, whereas systemic vasodilation does not. The systemic hemodynamic effects of SNOs occur after equilibration of the NO moiety amongst the plasma thiols via S-transnitrosylation.

Resveratrol Stimulates Hydrogen Sulfide (H2 S) Formation to Relax Murine Corpus Cavernosum.

INTRODUCTION: Resveratrol (RVT) found in red wine protects against erectile dysfunction and relaxes penile tissue (corpus cavernosum) via a nitric oxide (NO) independent pathway. However, the mechanism remains to be elucidated. Hydrogen sulfide (H2 S) is a potent vasodilator and neuromodulator generated in corpus cavernosum. AIMS: We investigated whether RVT caused the relaxation of mice corpus cavernosum (MCC) through H2 S. METHODS: H2 S formation is measured by methylene blue assay and vascular reactivity experiments have been performed by DMT strip myograph in CD1 MCC strips. MAIN OUTCOME MEASURES: Endothelial NO synthase (eNOS) inhibitor Nω-Nitro-L-arginine (L-NNA, 0.1 mM) or H2 S inhibitor aminooxyacetic acid (AOAA, 2 mM) which inhibits both cystathionine-ß-synthase (CBS) and cystathionine-gamma-lyase (CSE) enzyme or combination of AOAA with PAG (CSE inhibitor) has been used in the presence/absence of RVT (0.1 mM, 30 min) to elucidate the role of NO or H2 S pathways on the effects of RVT in MCC. Concentration-dependent relaxations to RVT, L-cysteine, sodium hydrogen sulfide (NaHS) and acetylcholine (ACh) were studied. RESULTS: Exposure of murine corpus cavernosum to RVT increased both basal and L-cysteine-stimulated H2 S formation. Both of these effects were reversed by AOAA but not by L-NNA. RVT caused concentration-dependent relaxation of MCC and that RVT-induced relaxation was significantly inhibited by AOAA or AOAA + PAG but not by L-NNA. L-cysteine caused concentration-dependent relaxations, which are inhibited by AOAA or AOAA + PAG significantly. Incubation of MCC with RVT significantly increased L-cysteine-induced relaxation, and this effect was inhibited by AOAA + PAG. However, RVT did not alter the effect of exogenous H2 S (NaHS) or ACh-induced relaxations. CONCLUSIONS: These results demonstrate that RVT-induced relaxation is at least partly dependent on H2 S formation and acts independent of eNOS pathway. In phosphodiesterase 5 inhibitor (PDE-5i) nonresponder population, combination therapy with RVT may reverse erectile dysfunction via stimulating endogenous H2 S formation.

Cysteinyl Leukotrienes (CysLTs): Role in Obesity-Induced Asthma.

Epidemiological studies associate obesity with onset of asthma, especially in obese children, suggesting obesity as the risk factor for asthma. Obesity-induced chronic inflammation has been implicated in the lung inflammation, yet specific mediators and mechanisms are lacking. Obesity is associated with increased expression of 5-lipoxygenase (5-LO) pathway and increased Leukotrienes (LTs) production has been observed in obese asthma patients. However, the precise mechanism that predisposes lungs inflammation in obese is not clearly understood. This article discusses the production and regulation of LTs in obese individuals and presents probable mechanisms regarding the role of LTs in lung inflammation that may lead to obesity-induced asthma. Leukotrienes are well known mediators of asthma but their role in obesity-induced asthma is not clearly understood and thus needs further research. Since efficient antagonists and inhibitors of 5-LO pathways are known, understanding of molecular mechanism of LTs, especially Cysteinyl-LTs, in obesity-induced asthma could lead to optimal treatment regimens for the prevention and treatment of asthma in obese individuals.