Tripeptide Glutathione Research Articles

A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity

SUMMARYThe tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38, leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity.

Anti-cancer organoruthenium(ii) complexes and their interactions with cysteine and its analogues. A mass-spectrometric study.

The ruthenium complexes [Ru(CYM)(p-Cl-dkt)(Cl)] (1), [Ru(CYM)(pta)(p-Cl-dkt)]PF6 (2), and [Ru(CYM)(pta)Cl2] (3, RAPTA-C) (CYM = para-cymene, p-Cl-dkt = 1-(4-chlorophenyl)-4,4,4-trifluorobutane-1,3-dione, pta = 1,3,5-triaza-7-phosphaadamantane) are biologically active and show anti-cancer activities, albeit with different mechanisms. To further understand these mechanisms, we compared their speciation in aqueous solutions with an amino acid (cysteine), with an amino acid derivative (N-acetylcysteine) and with a tripeptide (glutathione) by Mass Spectrometry (MS). Here, we show that all ruthenium complexes have high selectivity for cysteine and cysteine-derived molecules. On one hand, [Ru(CYM)(p-Cl-dkt)(Cl)] undergoes solvolysis in water and forms [Ru2(CYM)2(OH)3]+. Subsequently, all hydroxyl anions are exchanged by deprotonated cysteine. Infrared Photodissociation Spectroscopy (IRPD) showed that cysteine binds to the ruthenium atoms via the deprotonated thiol group and that sulfur bridges the ruthenium centers. On the other hand, the pta-bearing complexes remain monometallic and undergo only slow Cl or p-Cl-dkt exchange by deprotonated cysteine. Therefore, the pta ligand protects the ruthenium complexes from ligand exchange with water and from the formation of biruthenium clusters, possibly explaining why the mechanism of pta-bearing ruthenium complexes is not based on ROS production but on their reactivity as monometallic complexes.

Glutathione activation of an organometallic half-sandwich anticancer drug candidate by ligand attack.

In contrast to the clinical drug cisplatin, the anticancer complex [Os(η6-p-cymene)(4-(2-pyridylazo)-N,N-dimethylaniline)I]+ [1-I] is inert towards hydrolysis and targets cancer cell metabolism rather than DNA. A combination of DFT calculations and X-ray absorption spectroscopy (XAS) suggests that hydrolytic activation of 1-I involves catalytic attack by the intracellular tripeptide glutathione (GSH) on the azo bond of the chelating ligand in the complex.

New Variations on the Theme of Gold(III) C∧N∧N Cyclometalated Complexes as Anticancer Agents: Synthesis and Biological Characterization.

A series of novel (C∧N∧N) cyclometalated AuIII complexes of general formula [Au(bipydmb-H)X][PF6] (bipydmb-H = C∧N∧N cyclometalated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine) were prepared with a range of anionic ligands X in the fourth coordination position, featuring C (alkynyl)-, N-, O-, or S-donor atoms. The X ligands are varied in nature and include three coumarins, 4-ethynylaniline, saccharine, and thio-β-d-glucose tetraacetate, the tripeptide glutathione (GSH), and a coumarin-substituted amide derived from 4-ethynylaniline. The gold(I) complex [Au(C2ArNHCOQ)(PPh3)] (HC2ArNHCOQ = N-(4-ethynylphenyl)-2-oxo-2 H-chromene-3-carboxamide) was also prepared for comparison. The new compounds were fully characterized by means of analytical techniques, including NMR, absorption, and emission spectroscopy. The crystal structures of three cyclometalated AuIII complexes and of the AuI derivative were solved by single-crystal X-ray diffraction. The antiproliferative activity of the new AuIII cyclometalated derivatives was evaluated against cancer cells in vitro. According to the obtained results, only complexes 3-PF6 and 5-PF6, featuring coumarins as ancillary ligands and endowed with high redox stability in solution, display antiproliferative effects, with 5-PF6 being the most potent, while all of the others are scarcely active to nonactive in the selected cell lines. In order to study the reactivity of the compounds with biomolecules, the interaction of complexes 3-PF6 and 5-PF6 with the protein cytochrome c and the amino acids cysteine and histidine was analyzed by electrospray ionization mass spectrometry (ESI MS), showing adduct formation only with Cys after at least 1 h incubation. Furthermore, the parent hydroxo complex [Au(bipydmb-H)(OH)][PF6] (1OH-PF6) was investigated in a competitive assay to determine the protein vs oligonucleotide binding preferences by capillary zone electrophoresis (CZE) coupled to ESI-MS. Of note, the compound was found to selectively form adducts with the oligonucleotide over the protein upon ligand exchange with the hydroxido ligand. Adduct formation occurred within the first 10 min of incubation, demonstrating the preference of 1OH-PF6 for nucleotides in this setup. Overall, the obtained results point toward the possibility to selectively target DNA with gold(III) organometallics.

LC-MS/MS based detection and characterization of covalent glutathione modifications formed by reactive drug of abuse metabolites

Conjugation with the tripeptide glutathione (GSH) is a common mechanism of detoxification of many endogenous and exogenous compounds. This phenomenon typically occurs through the formation of a covalent bond between the nucleophilic free thiol moiety of GSH and an electrophilic site on the compound of interest.While GSH adducts have been identified for many licit drugs, there is a lack of information on the ability of drugs of abuse to adduct GSH. The present study utilized a metabolic assay with GSH as a nucleophilic trapping agent to bind reactive drug metabolites formed in situ.Extracted ion MS spectra were collected via LC-QqQ-MS/MS for all potentially significant ions and examined for fragmentation common to GSH-containing compounds, followed by confirmation of adduction and structural characterization performed by LC-QTOF-MS/MS.In addition to the two positive controls, of the 14 drugs of abuse tested, 10 exhibited GSH adduction, with several forming multiple adducts, resulting in a total of 22 individual identified adducts. A number of these are previously unreported in the literature, including those for diazepam, naltrexone, oxycodone and Δ9-THC.

Rigid dinuclear ruthenium-arene complexes showing strong DNA interactions

Six novel dinuclear Ru(II)-arene complexes [Ru2(η6-p-cymene)2(1,3-bib)2Cl2]×2·Solvent (X = Cl− (1), I− (2), NO3− (3), BF4− (4), PF6− (5), CF3SO3− (6); 1,3-bib = 1,3-di(1H-imidazol-1-yl) benzene) were synthesized and fully characterized by FT-IR, 1H NMR, ESI-MS, Elemental Analysis (EA) and Powder X-ray Diffraction (PXRD). Single crystal X-ray diffractions studies showed that 3 and 4 have rigid bowl-like structures, where one counter-anion (NO3− for 3 and BF4− for 4) was trapped inside the cavity to balance the charge, respectively. Even complexes 1–6 showed only moderate or little anti-proliferative activity toward cancer cells, strong interactions with DNA molecules through intercalation, however, were confirmed by UV-Vis, CD and fluorescence spectroscopy. Apoptosis and cell cycle arrest studies for complex 2 with cancer A549 cells indicated concentration-dependent late apoptosis and the G1/G0 phase arrest. Interactions with the tripeptide glutathione (γ-L-Glu-L-Cys-Gly, GSH) might explain the relatively low antiproliferative potency of these complexes. This class of rigid dinuclear cations hold potential as DNA-targeting anticancer agents if their uptake and delivery could be under controlled.

Nanoliter Sensing for Infrared Bioanalytics.

Nondestructive label-free bioanalytics of microliter to nanoliter sample volumes with low analyte concentrations requires novel analytic approaches. For this purpose, we present an optofluidic platform that combines surface-enhanced in situ infrared spectroscopy with microfluidics for sensing of surface-immobilized ultrathin biomolecular films in liquid analytes. Submonolayer sensitivity down to surface densities of few ng/cm2 is demonstrated for the adsorption of the thiolate tripeptide glutathione and for the recognition of streptavidin on a biotinylated enhancement substrate. Nonfunctionalized and functionalized metal island films on planar oxidized silicon substrates are used for signal enhancement with quantifiable enhancement properties. A single-reflection geometry at an incidence angle below the attenuated-total-reflection (ATR) regime is used with ordinary planar, IR-transparent windows. The geometry circumvents the strong IR absorption of common polymer materials and of aqueous environments in the IR fingerprint region. This practice enables straightforward quantitative analyses of, e.g., adsorption kinetics as well as chemical and structural properties in dependence of external stimuli.

Novel Dual Two‐Dimensional Liquid Chromatography Online Coupled to Ultraviolet Detector, Fluorescence Detector, Ion‐Trap Mass Spectrometer for Short Peptide Amino Acid Sequence Determination with Bottom‐Up Strategy

A novel dual two‐dimensional (2D) high‐performance liquid chromatography (LC) setup coupled online to an ultraviolet (UV) detector, fluorescence (FL) detector, and ion‐trap mass spectrometer (MS) has been developed for determining the amino acid sequence of short peptides using a novel bottom‐up strategy. Short peptides were electrothermally hydrolyzed to shorter peptides and amino acid enantiomers. The first 2D LC‐UV and FL system was used to separate and identify the produced parent and daughter short peptides and amino acid isomers and enantiomers in the hydrolysate; the second 2D LC‐MS was used to identify the presence of cysteine and obtain the molecular mass signals and N‐terminal peptide fragment ion signals for parent and daughter short peptides. The identified amino acid enantiomers are used to form any possible short peptides by permutation and combination in an order from dipeptide to a tripeptide, to a tetrapeptide, and to even higher short peptides. The correct short peptides are confirmed by comparing the molecular weights of the constituent amino acid enantiomers and the molecular weights of identified short peptides together, with the characteristic N‐terminal peptide fragment ion signals. The amino acid sequence of the dipeptide ester aspartame and the tripeptide glutathione was successfully determined by this method.

Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe-2S] cluster optical spectra and transfer chemistry.

Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions todisrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.

Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster

The tripeptide glutathione (GSH) is a crucial intracellular reductant and radical scavenger, but it may also coordinate the soft Cu(I) cation and thereby yield pro-oxidant species. The GSH-Cu(I) interaction is thus a key consideration for both redox and copper homeostasis in cells. However, even after nearly four decades of investigation, the nature and stability of the GSH-Cu(I) complexes formed under biologically relevant conditions remain controversial. Here, we revealed the unexpected predominance of a tetranuclear [Cu4(GS)6] cluster that is sufficiently stable to limit the effective free aquacopper(I) concentration to the sub-femtomolar regime. Combined spectrophotometric-potentiometric titrations at biologically realistic GSH/Cu(I) ratios, enabled by our recently developed Cu(I) affinity standards and corroborated by low-temperature phosphorescence studies, established cooperative assembly of [Cu4(GS)6] as the dominant species over a wide pH range, from 5.5 to 7.5. Our robust model for the glutathione-Cu(I) equilibrium system sets a firm upper limit on the thermodynamic availability of intracellular copper that is 3 orders of magnitude lower than previously estimated. Taking into account their ability to catalyze the production of deleterious superoxide, the formation of Cu(I)-glutathione complexes might be avoided under normal physiological conditions. The actual intracellular Cu(I) availability may thus be regulated a further 3 orders of magnitude below the GSH/Cu(I) affinity limit, consistent with the most recent affinity determinations of Cu(I) chaperones.

A Phi-Class Glutathione S-Transferase Gene for Verticillium Wilt Resistance in Gossypium arboreum Identified in a Genome-Wide Association Study.

Verticillium wilt disease is one of the most destructive biotic stresses faced by cotton plants. Here, we performed a genome-wide association study (GWAS) in 215 Chinese Gossypium arboreum accessions inoculated as seedlings with Verticillium dahliae to identify candidate loci involved in wilt resistance. We identified 309 loci that had a significant association with Verticillium wilt resistance and - log(P) values >5.0; the highest signal appeared on Ca3 in a 74 kb haplotype block. Five genes were also located within this haplotype block. One of these genes, CG05, was positioned close to the most significant SNP Ca3_23037225 (14 kb); expression of the gene was induced by V. dahliae or by treatment with salicylic acid (SA). Therefore, we suggest that CG05 may respond to invasion by V. dahliae via an SA-related signaling pathway, and we designated this gene as GaGSTF9. We showed that GaGSTF9 was a positive regulator of Verticillium wilt through the use of virus-induced gene silencing (VIGS) and overexpression in Arabidopsis. In addition, the glutathione S-transferase (GST) mutant gstf9 of Arabidopsis was found to be more susceptible to Verticillium wilt than wild-type plants. The levels of endogenous SA and hydrogen peroxide had a significant effect on Arabidopsis plants that overexpressed GaGSTF9, indicating that GST may regulate reactive oxygen species content via catalytic reduction of the tripeptide glutathione (GSH), and then affect SA content. Our data demonstrated that GaGSTF9 was a key regulator mediating cotton responses to V. dahliae and a potential candidate gene for cotton genetic improvement.

Synthesis of a water-soluble pillar[6]arene dodecaamine and its selective binding of acidic amino acids in water

A water-soluble pillar[6]arene dodecaamine has been synthesized. 1H NMR and fluorescence studies indicate that pillar[6]arene dodecaamine could selectively and strongly bind acidic amino acids, i.e. glutamic acid and aspartic acid in water. And the complexation behavior of pillar[6]arene dodecaamine towards acidic tripeptide glutathione and short chain length (C3 to C8) dicarboxylic acids in water is also investigated.

Mn2+-ZnSe/ZnS@SiO₂ Nanoparticles for Turn-on Luminescence Thiol Detection.

Biological thiols are antioxidants essential for the prevention of disease. For example, low levels of the tripeptide glutathione are associated with heart disease, cancer, and dementia. Mn2+-doped wide bandgap semiconductor nanocrystals exhibit luminescence and magnetic properties that make them attractive for bimodal imaging. We found that these nanocrystals and silica-encapsulated nanoparticle derivatives exhibit enhanced luminescence in the presence of thiols in both organic solvent and aqueous solution. The key to using these nanocrystals as sensors is control over their surfaces. The addition of a ZnS barrier layer or shell produces more stable nanocrystals that are isolated from their surroundings, and luminescence enhancement is only observed with thinner, intermediate shells. Tunability is demonstrated with dodecanethiol and sensitivities decrease with thin, medium, and thick shells. Turn-on nanoprobe luminescence is also generated by several biological thiols, including glutathione, N-acetylcysteine, cysteine, and dithiothreitol. Nanoparticles prepared with different ZnS shell thicknesses demonstrated varying sensitivity to glutathione, which allows for the tuning of particle sensitivity without optimization. The small photoluminescence response to control amino acids and salts indicates selectivity for thiols. Preliminary magnetic measurements highlight the challenge of optimizing sensors for different imaging modalities. In this work, we assess the prospects of using these nanoparticles as luminescent turn-on thiol sensors and for MRI.

A novel hydrophilic interaction liquid chromatography method for the determination of underivatized amino acids in alimentary supplements

Amino acids playing important roles in metabolic processes are often included in dietary supplements whose use has largely expanded over the last 20 years not only in patients with particular deficiencies, but also in athletes and even common people that want to enrich their regular daily diet. In the present study, a bare silica Kinetex core-shell 2.6μm HILIC column was used for separation of some important hydrophilic amino acids and amino acids-like molecules i.e., aspartic acid, creatine, carnitine, arginine and the tripeptide glutathione (GSH), by optimizing the chromatographic conditions for their determination in complex alimentary supplements. The contribution of partition, adsorption and ion exchange on the retention mechanism was studied by varying parameters such as water content and the counter-ion concentration in the mobile phase. Optimum conditions employed a Phenomenex Kinetex core-shell 2.6μm HILIC (100×4.6mm i.d.) column and a mobile phase of acetonitrile/potassium phosphate buffer (12.5mM; pH=2.8) 85:15, v/v, at the flow rate of 1.4mL/min, using UV detection at 200nm. A reference HPLC method for the selective determination of GSH by using 1,4-naphthoquinone as derivatization reagent was also introduced for comparative purposes.The developed HILIC method was validated and applied to the analysis of the considered compounds in dietary supplements. Interestingly, in some of the real samples, oxidized glutathione which is an inactive impurity of GSH, was found at the level of about 20%. The proposed study confirms the importance of simple analytical methods for a rigorous quality control of dietary supplements containing unstable active ingredients.

Quantitation of glutathione S-transferases in rice (Oryza sativa L.) roots exposed to cadmium by liquid chromatography\u2013tandem mass spectrometry using isotope-labeled wing peptides as an internal standard

BackgroundPlant glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional enzymes involved in heavy metal cellular detoxification by conjugating the tripeptide (g-Glu-Cys-Gly) glutathione to heavy metals. Previous studies demonstrated that individual rice GSTs were differentially induced by heavy metal exposure at the mRNA transcript level. However, little information is available concerning changes in protein concentration of rice GSTs under heavy metal stress. Because the correlation between changes in protein concentration and gene expression under abiotic stress is poor, direct determination of rice GSTs protein concentrations during cadmium (Cd) exposure is a more effective and reliable approach to explore possible mechanisms of rice Cd translocation and accumulation.ResultsThis study established an optimized and advanced liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based targeted proteomics assay for quantification of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots. The tryptic signature peptides were chosen as surrogate analytes and winged peptides containing the isotope-labeled signature peptides were used as the internal standards. The signature peptides exhibited good linearity in the range of 0.6–60 and 0.3–30 nM, respectively. The limit of detection and limit of quantification were 4.5 and 14.5 µg/g for OsGSTF14, respectively, and 2.1 and 7.0 µg/g for OsGSTU6. The spiking recoveries rates at low, medium and high levels were in the range of 72.5–93.4%, with intra- and inter-day precisions of 5.5–9.1 and 4.2–10.2%, respectively.ConclusionsThe assay successfully quantified the temporal and dose responses of OsGSTF14 and OsGSTU6 proteins in Cd-stressed rice roots, with good accuracy, precision and high-throughput. This assay will have significant application in developing quantification methods of other proteins in Cd-stressed rice, which may provide more insight into the mechanisms of Cd translocation and accumulation in rice.

Analytical and structural investigation via infrared spectroscopy and density functional methods of cuprous complexes of the antioxidant tripeptide glutathione (GSH). Synthesis and characterization of a novel CuI-GSH compound

A copper(I) compound of reduced glutathione (Glu,S;Cys,R)-(GSH) was isolated from the reaction of CuICl and zwitterionic H3GSH(+−) in acetonitrile:water medium. The analytical data are in agreement with the formula CuI(H2GSH)(H2O)2. The characterization was carried out via infrared spectroscopy and density functional (DFT) method computations, that allowed to fully optimize structures for the free ligands and for several complex molecules of the type [CuI(S,O,O-H2GSH)(OH2)] having a pseudo-tetrahedral coordination arrangement and the Gly residue deprotonated at carboxylic group. The molecules were fully optimized at DFT-B3LYP/6-31G∗∗ (BS1) and in some cases at B3LYP/6-311++G∗∗ (BS2) and the solvent treatment (water, via PCM method) was taken into account (BS1W). The comparative analyses between experimental and computed infrared spectra confirmed that the free ligand was reduced glutathione in the zwitterionic form at the Glu residue. The analysis showed also that the metal compound consisted of complex molecules of the type [CuI(S,O,O-H2GSH(Gly))str(H2O)] where the ligand had a stretched (str) arrangement assisted by a network of hydrogen bonds. The presence of a globular (glo) structure for the complex molecule that brought the Glu-COOH and Glu-NH2 groups towards the Cys-SH and the Gly-COO− donors, respectively, could not be ruled out.

Assessing the risk of cytomegalovirus DNAaemia in allogeneic stem cell transplant recipients by monitoring oxidative-stress markers in plasma.

The level of antioxidants, such as thiol-containing tripeptide glutathione (GSH), in cytomegalovirus (CMV)-infected cells is notably increased. We previously showed that GSH levels in plasma, as measured by untargeted 1H nuclear magnetic resonance, are higher in allogeneic stem cell transplant (allo-SCT) recipients who subsequently develop CMV viraemia. We hypothesized that the net level of oxidative-stress markers present in plasma may be reduced in patients who develop CMV DNAaemia compared to those who do not. We serially monitored the levels of malondialdehyde (MDA) and carbonylated proteins (CPs) early after allo-SCT and assessed whether they could predict the occurrence of CMV DNAaemia. MDA levels were measured in 43 patients (28 had CMV DNAaemia) and CPs were quantified in 53 patients (38 patients developed CMV DNAaemia). The area under the curve (AUC) value for MDA, but not for CPs, was significantly lower in patients who subsequently developed CMV DNAaemia compared to those who remained DNAaemia-free (P=0.043). A trend toward lower MDA AUC values was observed in episodes of CMV DNAaemia with faster CMV replicative kinetics and in those who reached higher peak CMV DNA levels. Moreover, receiver operating characteristic curve analyses indicated that the MDA biomarker had the predictive ability to discriminate between patients with or without subsequent CMV DNAaemia (AUC=0.69, 95 % confidence interval 0.51-0.85, P=0.05). In summary, serial quantitation of MDA may be useful for individualizing antiviral prophylaxis therapies (targeted prophylaxis) in the upcoming era of new antiviral drugs with improved safety profiles.

Abstract 105: Depletion of extracellular cystine and cysteine by a mutagenized human enzyme causes ROS mediated cytotoxicity in pancreatic cancer cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate at 7%. The oncogene KRAS that is mutationally activated in over 90% of PDACs, has been shown to be involved in regulating redox homeostasis. Hence, perturbation of oxidative balance might provide a therapeutic window to effectively treat pancreatic cancer. One strategy to achieve this is to target the tripeptide glutathione (GSH) – a major intracellular antioxidant. Cysteine (Cys), which has the functional moiety of GSH, can either be synthesized de novo or imported, predominantly as cystine (CSSC) that is then reduced intracellularly to Cys. In cancer, intracellular Cys synthesis has to be supplemented with extracellular import in order to fulfil the excessive metabolic demand of proliferation, which includes maintenance of oxidative balance through GSH synthesis. We hypothesize that this increased requirement for Cys/CSSC import in tumor cells will make them selectively sensitive to prolonged depletion of these amino acids in the serum by a genetically engineered human enzyme called Cyst(e)inase. In addition, we believe that combining Cyst(e)inase with other redox balance perturbing agents will produce a synergistic therapeutic effect. In our study, Cyst(e)inase treatment of cultured pancreatic cancer cell lines (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Sensitivity to Cyst(e)inase was correlated with ROS accumulation (Panc1>MIA-PaCa2>BxPC3). Panc1 cells exhibited G2-arrest and apoptotic cell death following 24 hours of treatment whereas BxPC3 cells underwent only a G1-arrest and no cell death even after 72 hours of treatment. Further mechanistic investigation showed activation of AMP kinase and other stress related kinases (p38, ERK and JNK), and DNA damage signaling (ATM) only in the more sensitive cell lines. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway was observed in all 3 cell lines. Cyst(e)inase displayed synergistic cytotoxicity when combined with buthionine sulfoximine (GSH synthesis inhibitor), auranofin (thioredoxin reductase inhibitor), sulfasalazine (inhibitor of cystine import) and the natural compound curcumin known to increase intracellular ROS, indicating that concurrently inhibiting alternative cellular antioxidant pathways or directly increasing intracellular ROS might improve the anti-tumor efficacy of Cyst(e)inase. The effect of biweekly intraperitoneal Cyst(e)inase treatment on growth of pancreatic cancer cell xenografts in nude mice will also be reported. Collectively, the current data suggest that depletion of extracellular Cys/CSSC using Cyst(e)inase may have utility either as a monotherapy or a combination therapy for pancreatic cancer. Citation Format: Sabin Kshattry, Achinto Saha, Shira Cramer, John DiGiovanni, Stefano Tiziani, Nathalie Munoz, George Georgiou, Everett Stone. Depletion of extracellular cystine and cysteine by a mutagenized human enzyme causes ROS mediated cytotoxicity in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 105. doi:10.1158/1538-7445.AM2017-105

Effect of Microwave Heating on the Crystallization of Glutathione Tripeptide on Silver Nanoparticle Films.

Effect of microwave heating on the crystallization of glutathione (GSH) tripeptide using the metal-assisted and microwave-accelerated evaporative crystallization (MA-MAEC) technique is reported. GSH crystals were grown from supersaturated solutions of GSH (300-500 mg/mL) on the iCrystal plates with silver nanoparticle films (SNFs) and without SNFs in three different microwave systems operating at 2.45 GHz: conventional (multimode, fixed power at 900W), industrial (monomode, variable power up to 1200 W), and the iCrystal system (monomode, variable power up to 100 W). The efficacy of the MA-MAEC technique, in terms of improvement in the crystallization time, crystal size and quality of GSH, was compared between the three microwave systems and the crystallization at room temperature (no microwave heating, a control experiment). Optical microscopy was used to visualize and quantify the growth of GSH crystals during and after microwave heating. Powder X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy data showed that GSH crystals had identical crystal structure to those grown at room temperature and microwave heating did not alter the chemical structure of GSH molecules during microwave heating, respectively. Using the MA-MAEC technique, the iCrystal system yielded high quality GSH crystals in a rapid manner.

Glutathione and Glutathione Transferase Omega 1 as Key Posttranslational Regulators in Macrophages.

Macrophage activation during phagocytosis or by pattern recognition receptors, such as Toll-like receptor 4, leads to the accumulation of reactive oxygen species (ROS). ROS act as a microbicidal defense mechanism, promoting clearance of infection, allowing for resolution of inflammation. Overproduction of ROS, however, overwhelms our cellular antioxidant defense system, promoting oxidation of protein machinery, leading to macrophage dysregulation and pathophysiology of chronic inflammatory conditions, such as atherosclerosis. Here we will describe the role of the antioxidant tripeptide glutathione (GSH). Until recently, the binding of GSH, termed glutathionylation, was only considered to maintain the integrity of cellular components, limiting the damaging effects of an aberrant oxidative environment. GSH can, however, have positive and negative regulatory effects on protein function in macrophages. GSH regulates protein secretion, driving tumor necrosis factor α release, hypoxia-inducible factor-1α stability, STAT3 phosphorylation, and caspase-1 activation in macrophages. GSH also plays a role in host defense against Listeria monocytogenes, modifying the key virulence protein PrfA in infected macrophages. We will also discuss glutathione transferase omega 1, a deglutathionylating enzyme recently shown to play a role in many aspects of macrophage activity, including metabolism, NF-κB activation, and cell survival pathways. Glutathionylation is emerging as a key regulatory event in macrophage biology that might be susceptible to therapeutic targeting.