We report here the development and optimization of a simple 384-well colorimetric assay to measure H2O2 generated by the redox cycling of compounds incubated with reducing agents in high-throughput screening (HTS) assay buffers. The phenol red-horseradish peroxidase (HRP) assay readily detected H2O2 either added exogenously or generated by the redox cycling of compounds in dithiothreitol (DTT). The generation of H2O2 was dependent on the concentration of both the compound and DTT and was abolished by catalase. Although both DTT and tris(2-carboxyethyl)-phosphine sustain the redox cycling generation of H2O2 by a model quinolinedione, 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione (NSC 663284; DA3003-1), other reducing agents such as β-mercaptoethanol, glutathione, and cysteine do not. The assay is compatible with HTS. Once terminated, the assay signal was stable for at least 5 h, allowing for a reasonable throughput. The assay tolerated up to 20% dimethyl sulfoxide, allowing a wide range of compound concentrations to be tested. The assay signal window was robust and reproducible with average Z-factors of ≥0.8, and the redox cycling generation of H2O2 by DA3003-1 in DTT exhibited an average 50% effective concentration of 0.830 μ 0.068 μM. Five of the mitogen-activated protein kinase phosphatase (MKP) 1 inhibitors identified in an HTS were shown to generate H2O2 in the presence of DTT, and their inhibition of MKP-1 activity was shown to be time dependent and was abolished or significantly reduced by either 100 U of catalase or by higher DTT levels. A cross-target query of the PubChem database with three structurally related pyrimidotriazinediones revealed active flags in 36–39% of the primary screening assays. Activity was confirmed against a number of targets containing active site cysteines, including protein tyrosine phosphatases, cathepsins, and caspases, as well as a number of cellular cytotoxicity assays. Rather than utilize resources to conduct a hit characterization effort involving several secondary assays, the phenol red-HRP assay provides a simple, rapid, sensitive, and inexpensive method to identify compounds that redox cycle in DTT or tris(2-carboxyethyl)phosphine to produce H2O2 that may indirectly modulate target activity and represent promiscuous false-positives from a primary screen.
We have screened the Library of Pharmacologically Active Compounds (LOPAC) and the National Institutes of Health (NIH) Small Molecule Repository (SMR) libraries in a horseradish peroxidase–phenol red (HRP-PR) H2O2 detection assay to identify redox cycling compounds (RCCs) capable of generating H2O2 in buffers containing dithiothreitol (DTT). Two RCCs were identified in the LOPAC set, the ortho-naphthoquinone β-lapachone and the para-naphthoquinone NSC 95397. Thirty-seven (0.02%) concentration-dependent RCCs were identified from 195,826 compounds in the NIH SMR library; 3 singleton structures, 9 ortho-quinones, 2 para-quinones, 4 pyrimidotriazinediones, 15 arylsulfonamides, 2 nitrothiophene-2-carboxylates, and 2 tolyl hydrazides. Sixty percent of the ortho-quinones and 80% of the pyrimidotriazinediones in the library were confirmed as RCCs. In contrast, only 3.9% of the para-quinones were confirmed as RCCs. Fifteen of the 251 arylsulfonamides in the library were confirmed as RCCs, and since we screened 17,868 compounds with a sulfonamide functional group we conclude that the redox cycling activity of the arylsulfonamide RCCs is due to peripheral reactive enone, aromatic, or heterocyclic functions. Cross-target queries of the University of Pittsburgh Drug Discovery Institute (UPDDI) and PubChem databases revealed that the RCCs exhibited promiscuous bioactivity profiles and have populated both screening databases with significantly higher numbers of active flags than non-RCCs. RCCs were promiscuously active against protein targets known to be susceptible to oxidation, but were also active in cell growth inhibition assays, and against other targets thought to be insensitive to oxidation. Profiling compound libraries or the hits from screening campaigns in the HRP-PR H2O2 detection assay significantly reduce the timelines and resources required to identify and eliminate promiscuous nuisance RCCs from the candidates for lead optimization.
Cdc25 phosphatases are dual-specific phosphatases that play a role in cell cycle progression. In many human cancers, Cdc25 phosphatases are overexpressed as compared with normal tissues. In addition, overexpression of Cdc25 phosphatases in prostate cancer is correlated with disease progression. The antiproliferative efficacy of Cdc25 phosphatase inhibitor 7-chloro-6-(2-morpholin-4-ylethylamino) quinoline-5, 8-dione (DA 3003-2) was investigated in the PC-3 asynchronous human prostate cancer cell line using a cell-based assay. The time course changes in cell cycle distribution and the modulation of cell cycle regulators after DA 3003-2 administration were analyzed using the MTT assay. We found that the relative IC50 of DA 3003-2 was 2-fold lower as compared with its congener (2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone (NSC 672121). Asynchronous PC-3 cells accumulated in the G2/M phase at 24 h after treatment with 10 μM DA 3003-2 or 20 μM NSC 672121, which represent IC70 concentrations. Treatment of cells with DA 3003-2 caused hyperphosphorylation of Cdc2 tyr15 in cyclin B1 and cyclin A complexes. DA 3003-2 did not downregulate the protein expression levels of Cdc25s, cyclins and cyclin-dependent kinases (Cdks). To conclude, after DA 3003-2 administration asynchronous PC-3 cells accumulated in the G2/M phase, with hyperphosphorylation of the G2/M cyclin-Cdk complex.
prostate cancer; Cdc25 phosphatase; cell cycle; DA3003-2; 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5; 8-dione; small molecular target
High-throughput screening (HTS) of large compound libraries has become a commonly used method for the identification of drug leads, and non-physiological reducing agents have been widely used for HTS. However, a comparison of the difference in the HTS results based on the choice of reducing agent used and potency comparisons of selected inhibitors has not been done with the physiological reducing agent, reduced glutathione (GSH). Here, we compared the effects of three reducing agents: dithiothreitol (DTT), β-mercaptoethanol (β-MCE), and tris-(2-carboxyethyl)-phosphine (TCEP), in addition to GSH, against three drug target proteins. Approximately 100,000 compounds were computationally screened for each target protein, and experimental testing of high scoring compounds (~560 compounds) with the four reducing agents surprisingly produced many non-overlapping hits. More importantly, we find that various reducing agents alter inhibitor potency (IC50) from ~10 µM with one reducing agent to complete loss (IC50 > 200 µM) of inhibitory activity with another reducing agent. Therefore, the choice of reducing agent in a HTS is critical as this may lead to the pursuit of falsely identified active compounds or failure to identify the true active compounds. We demonstrate the feasibility of using GSH for in vitro HTS assays with these three target enzymes.
Reducing agent; inhibitor screening; false positives; false negatives; potency comparison
A redox-responsive polycationic system was synthesized via copolymerization of N,N-diethylacrylamide (DEAAm) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). N,N’-bis(4-chlorobutanoyl)cystamine was used as disulfide-containing cross-linker to form networks by the quaternization of tertiary amine groups. The insoluble cationic hydrogels become soluble by reduction of disulfide to mercaptanes by use of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) or cysteamine, respectively. The soluble polymeric system can be cross-linked again by using oxygen or hydrogen peroxide under basic conditions. The redox-responsive polymer networks can be used for molecular inclusion and controlled release. As an example, phenolphthalein, methylene blue and reactive orange 16 were included into the network. After treatment with DTT a release of the dye could be recognized. Physical properties of the cross-linked materials, e.g., glass transition temperature (T
g), swelling behavior and cloud points (T
c) were investigated. Redox-responsive behavior was further analyzed by rheological measurements.
cationic hydrogel; cross-linked polymer; 2-(dimethylamino)ethyl methacrylate (DMAEMA); disulfide cleavage; N,N-diethylacrylamide (DEAAm)
SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.
The rate of consumption of dithiothreitol (DTT) is increasingly used to measure the oxidative potential of particulate matter (PM), which has been linked to the adverse health effects of PM. While several quinones are known to be very reactive in the DTT assay, it is unclear what other chemical species might contribute to the loss of DTT in PM extracts. To address this question, we quantify the rate of DTT loss from individual redox-active species that are common in ambient particulate matter. While most past research has indicated that the DTT assay is not sensitive to metals, our results show that seven out of the ten transition metals tested do oxidize DTT, as do three out of the five quinones tested. While metals are less efficient at oxidizing DTT compared to the most reactive quinones, concentrations of soluble transition metals in fine particulate matter are generally much higher than those of quinones. The net result is that metals appear to dominate the DTT response for typical ambient PM2.5 samples. Based on particulate concentrations of quinones and soluble metals from the literature, and our measured DTT responses for these species, we estimate that for typical PM2.5 samples approximately 80 % of DTT loss is from transition metals (especially copper and manganese), while quinones account for approximately 20 %. We find a similar result for DTT loss measured in a small set of PM2.5 samples from the San Joaquin Valley of California. Because of the important contribution from metals, we also tested how the DTT assay is affected by EDTA, a chelator that is sometimes used in the assay. EDTA significantly suppresses the response from both metals and quinones; we therefore recommend that EDTA should not be included in the DTT assay.
The contribution of the N-methyl D-aspartate receptors (NMDARs) to synaptic plasticity declines during aging and the decline is thought to contribute to memory deficits. Here, we demonstrate that an age-related shift in intracellular redox state contributes to the decline in NMDAR responses through Ca2+/calmodulin-dependent protein kinase II (CaMKII). The oxidizing agent xanthine/xanthine oxidase (X/XO) decreased the NMDAR mediated synaptic responses at hippocampal CA3-CA1 synapses in slices from young (3–8 mo), but not aged (20–25 mo) rats. Conversely, the reducing agent dithiothreitol (DTT) selectively enhanced NMDAR response to a greater extent in aged hippocampal slices. The enhancement of NMDAR responses facilitated induction of long-term potentiation (LTP) in aged but not young animals. The DTT-mediated growth in the NMDAR response was not observed for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) mediated synaptic responses. A similar increase was observed by intracellular application of the membrane impermeable reducing agent, L-glutathione (L-GSH), through the intracellular recording pipette, indicating the increased NMDAR response was dependent on intracellular redox state. DTT enhancement of the NMDAR response was dependent on CaMKII activity and was blocked by the CaMKII inhibitor – myristoylated autocamtide-2 related inhibitory peptide (myr-AIP), but not by inhibition of the activity of protein phosphatases - PP1 and calcineurin (CaN/PP2B) or protein kinase C. CaMKII activity assays established that DTT increased CaMKII activity in CA1 cytosolic extracts in aged but not in young animals. These findings indicate a link between oxidation of CaMKII during aging, a decline in NMDAR responses, and altered synaptic plasticity.
NMDA receptor; aging; hippocampus; ROS; oxidative stress; CaMKII; long-term potentiation
Typically, simple flavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen peroxide without release of significant levels of the superoxide ion. However, two evolutionarily-related single-domain sulfhydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predominantly found in the mitochondrial intermembrane) release up to ~30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofactor within the helix-rich Erv domain. Subsequent reduction of the flavin is followed by transfer of reducing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (THP) as an alternative reducing substrate to dithiothreitol (DTT). THP, and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the formation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP) respectively. This superoxide-dependent amplification of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemiluminescence method using a luciferin analog (MCLA) when 2 mM DTT was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~65% when monomeric mutants of the normally homodimeric enzyme were used. In contrast, monomeric multi-domain Quiescin-sulfhydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed.
The growing body of clinical and experimental data regarding electromagnetic field (EMF) bioeffects and their therapeutic applications has contributed to a better understanding of the underlying mechanisms of action. This study reports that two EMF modalities currently in clinical use, a pulse-modulated radiofrequency (PRF) signal, and a static magnetic field (SMF), applied independently, increased the rate of deoxygenation of human hemoglobin (Hb) in a cell-free assay. Deoxygenation of Hb was initiated using the reducing agent dithiothreitol (DTT) in an assay that allowed the time for deoxygenation to be controlled (from several min to several hours) by adjusting the relative concentrations of DTT and Hb. The time course of Hb deoxygenation was observed using visible light spectroscopy. Exposure for 10–30 min to either PRF or SMF increased the rate of deoxygenation occurring several min to several hours after the end of EMF exposure. The sensitivity and biochemical simplicity of the assay developed here suggest a new research tool that may help to further the understanding of basic biophysical EMF transduction mechanisms. If the results of this study were to be shown to occur at the cellular and tissue level, EMF-enhanced oxygen availability would be one of the mechanisms by which clinically relevant EMF-mediated enhancement of growth and repair processes could occur.
The translocation mode of preprolactin (pPL) across mammalian endoplasmic reticulum was reinvestigated in light of recent findings that nascent secretory polypeptides synthesized in the presence of a highly reducing environment could be translocated posttranslationally and independently of their attachment to the ribosome (Maher, P. A., and S. J. Singer, 1986, Proc. Natl. Acad. Sci. USA, 83:9001-9005). The effects of the reducing agent dithiothreitol (DTT) on pPL synthesis and translocation were studied in this respect. The translocation of pPL was shown to take place only cotranslationally. The apparent posttranslational translocation was due to ongoing chain synthesis irrespective of the presence of high concentrations of DTT. When synthesis was completely blocked, no translocation was observed in the presence or absence of DTT. The synthesis of pPL was retarded by DTT, while its percent translocation was enhanced. The retardation in synthesis was reflected in reduced rates of initiation and elongation. As a consequence of this retardation, which increases the ratio of microsomes to nascent chains, and of possible effects on the conformation of nascent pPL and components of the translocation apparatus, DTT may expand the time and chain length windows for nascent chain translocation competence.
Particulate matter (PM) air pollution has a significant impact on human morbidity and mortality; however, the mechanisms of PM-induced toxicity are poorly defined. A leading hypothesis states that airborne PM induces harm by generating reactive oxygen species (ROS) in and around human tissues, leading to oxidative stress. We report here, a system employing a microfluidic electrochemical sensor coupled directly to a Particle-into-Liquid-Sampler (PILS) system to measure aerosol oxidative activity in an on-line format. The oxidative activity measurement is based on the dithiothreitol assay (DTT assay) where after oxidized by PM, the remaining reduced DTT was analyzed by the microfluidic sensor. The sensor consists of an array of working, reference, and auxiliary electrodes fabricated in a poly(dimethylsiloxane) (PDMS)-based microfluidic device. Cobalt (II) phthalocyanine (CoPC)-modified carbon paste was used as the working electrode material allowing selective detection of reduced DTT. The electrochemical sensor was validated off-line against the traditional DTT assay using filter samples taken from urban environments and biomass burning events. After off-line characterization, the sensor was coupled to a PILS to enable on-line sampling/analysis of aerosol oxidative activity. Urban dust and industrial incinerator ash samples were aerosolized in an aerosol chamber and analyzed for their oxidative activity. The on-line sensor reported DTT consumption rates (oxidative activity) in good correlation with aerosol concentration (R2 from 0.86–.97) with a time-resolution of approximately 3 minutes.
Redox cycling compounds (RCCs) generate µM concentrations of hydrogen peroxide (H2O2) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H2O2 generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H2O2-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H2O2 in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H2O2 as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds which can generate intracellular H2O2 by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; critical resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs.
Free nitric oxide (NO) reacts with sulphydryl residues to form S-nitrosothiols, which act as NO reservoirs. We sought to determine whether thiol-preserving agents and antioxidants, such as dithiothreitol (DTT) and vitamin C, induce NO release from S-nitrosylated proteins in endothelial cell cultures to promote angiogenesis.
NO release was measured directly in cell supernatants using a Sievers NO Analyser, and in vitro angiogenesis was assessed by quantifying capillary-like tube network formation of porcine aortic endothelial cells (PAEC) on growth factor-reduced Matrigel. Incubation of PAEC with DTT or vitamin C significantly increased NO release in a concentration-dependent manner. However, the nitric oxide synthase (NOS) inhibitors, L-NNA and L-NIO, had no effect on DTT- or vitamin C-induced NO release, and there was no concomitant increase in the phosphorylation of endothelial NOS at serine-1177 following DTT or vitamin C treatment. DTT and vitamin C increased capillary-like tube network formation by nine- and two-fold, respectively, and the addition of copper ions doubled the effect of vitamin C. Surprisingly, DTT maintained endothelial tube networks for up to one month under serum-free conditions, and selective inhibitors of guanylyl cyclase (ODQ) and PKG (KT-5823) blocked this, demonstrating the requirement of cyclic GMP and PKG in this process.
Both DTT and vitamin C are capable of releasing sufficient NO from S-nitrosothiols to induce capillary morphogenesis. This study provides the first evidence that increased denitrosylation leads to increased bioavailability of NO, independent of NOS activity, to promote sustained angiogenesis.
Rotavirus undergoes a unique mode of assembly in the rough endoplasmic reticulum (RER) of infected cells. Luminal RER proteins undergo significant cotranslational and posttranslational modifications, including disulfide bond formation. Addition of a reducing agent (dithiothreitol [DTT]) to rotavirus-infected cells did not significantly inhibit translation or disrupt established disulfide bonds in rotavirus proteins but prevented the formation of new disulfide bonds and infectious viral progeny. In DTT-treated, rotavirus-infected cells, all vp4, vp6, and ns28 epitopes but no vp7 epitopes were detected by immunohistochemical staining with a panel of monoclonal antibodies. When oxidizing conditions were reestablished in DTT-treated cells, intramolecular disulfide bonds in vp7 were rapidly and correctly established with the restoration of antigenicity, although prolonged DTT treatment led to the accumulation of permanently misfolded vp7. Electron microscopy revealed that cytosolic assembly of single-shelled particles and budding into the ER was not affected by DTT treatment but that outer capsid assembly was blocked, leading to the accumulation of single-shelled and enveloped intermediate subviral particles in the RER lumen.
Substituted pyrazole esters were identified as hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the enzyme cathepsin B. Members of this class, along with functional group analogs, were synthesized in an effort to define the structural requirements for activity. Analog characterization was hampered by the need to include a reducing agent such as dithiothreitol (DTT) or cysteine in the assay, highlighting the caution required in interpreting biological data gathered in the presence of such nucleophiles. Despite the confounding effects of DTT and cysteine, our studies demonstrate that the pyrazole 1 acts as alternate substrate for cathepsin B, rather than as an inhibitor.
We report the first detailed investigation of the kinetics
splicing by the Methanococcus jannaschii KlbA (Mja KlbA) intein. This intein has an N-terminal Ala in place
of the nucleophilic Cys or Ser residue that normally initiates splicing
but nevertheless splices efficiently in vivo [Southworth, M. W., Benner,
J., and Perler, F. B. (2000) EMBO J.19, 5019–5026]. To date, the spontaneous nature of the cis splicing
reaction has hindered its examination in vitro. For this reason, we
constructed an Mja KlbA intein–mini-extein
precursor using intein-mediated protein ligation and engineered a
disulfide redox switch that permits initiation of the splicing reaction
by the addition of a reducing agent such as dithiothreitol (DTT).
A fluorescent tag at the C-terminus of the C-extein permits monitoring
of the progress of the reaction. Kinetic analysis of the splicing
reaction of the wild-type precursor (with no substitutions in known
nucleophiles or assisting groups) at various DTT concentrations shows
that formation of the branched intermediate from the precursor is
reversible (forward rate constant of 1.5 × 10–3 s–1 and reverse rate constant of 1.7 × 10–5 s–1 at 42 °C), whereas the
productive decay of this intermediate to form the ligated exteins
is faster and occurs with a rate constant of 2.2 × 10–3 s–1. This finding conflicts with reports about
standard inteins, for which Asn cyclization has been assigned as the
rate-determining step of the splicing reaction. Despite being the
slowest step of the reaction, branched intermediate formation in the Mja KlbA intein is efficient in comparison with those of
other intein systems. Interestingly, it also appears that this intermediate
is protected against thiolysis by DTT, in contrast to other inteins.
Evidence is presented in support of a tight coupling between the N-terminal
and C-terminal cleavage steps, despite the fact that the C-terminal
single-cleavage reaction occurs in variant Mja KlbA
inteins in the absence of N-terminal cleavage. We posit that the splicing
events in the Mja KlbA system are tightly coordinated
by a network of intra- and interdomain noncovalent interactions, rendering
its function particularly sensitive to minor disruptions in the intein
or extein environments.
Using recombinant DNA technology for expression of protein therapeutics is a maturing field of pharmaceutical research and development. As recombinant proteins are increasingly utilized as biotherapeutics, improved methodologies ensuring the characterization of post-translational modifications (PTMs) are needed. Typically, proteins prepared for PTM analysis are proteolytically digested and analyzed by mass spectrometry. To assure full coverage of the PTMs on a given protein, one must obtain complete sequence coverage of the protein, which is often quite challenging. The objective of the research described here is to design a protocol that maximizes protein sequence coverage and enables detection of post-translational modifications, specifically N-linked glycosylation. To achieve this objective, a highly efficient proteolytic digest protocol using trypsin was designed by comparing the relative merits of denaturing agents (urea and Rapigest™ SF), reducing agents (dithiothreitol, DTT, and tris(2-carboxyethyl)phophine, TCEP), and various concentrations of alkylating agent (iodoacetamide, IAM). After analysis of human apo-transferrin using various protease digestion protocols, ideal conditions were determined to contain 6 M urea for denaturation, 5 mM TCEP for reduction, 10 mM IAM for alkylation, and 10 mM DTT, to quench excess IAM before the addition of trypsin. This method was successfully applied to a novel recombinant protein, human lysyl oxidase-like 2 (hLOXL2). Furthermore, the glycosylation PTMs were readily detected at two glycosylation sites in the protein. These digestion conditions were specifically designed for PTM analysis of recombinant proteins and biotherapeutics, and the work described herein fills an unmet need in the growing field of biopharmaceutical analysis.
We previously showed that thioredoxins are required for dithiothreitol (DTT) tolerance, suggesting they maintain redox homeostasis in response to both oxidative and reductive stress conditions. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to DTT to identify cell functions involved in resistance to reductive stress. We identified 195 mutants, whose gene products are localized throughout the cell. DTT-sensitive mutants were distributed among most major biological processes, but they particularly affected gene expression, metabolism, and the secretory pathway. Strikingly, a mutant lacking TSA1, encoding a peroxiredoxin, showed a similar sensitivity to DTT as a thioredoxin mutant. Epistasis analysis indicated that thioredoxins function upstream of Tsa1 in providing tolerance to DTT. Our data show that the chaperone function of Tsa1, rather than its peroxidase function, is required for this activity. Cells lacking TSA1 were found to accumulate aggregated proteins, and this was exacerbated by exposure to DTT. Analysis of the protein aggregates revealed that they are predominantly composed of ribosomal proteins. Furthermore, aggregation was found to correlate with an inhibition of translation initiation. We propose that Tsa1 normally functions to chaperone misassembled ribosomal proteins, preventing the toxicity that arises from their aggregation.
Thiol-disulfide bond balance is generally maintained in bacteria by thioredoxin reductase-thioredoxin and/or glutathione-glutaredoxin systems. Some gram-positive bacteria, including Lactococcus lactis, do not produce glutathione, and the thioredoxin system is presumed to be essential. We constructed an L. lactis trxB1 mutant. The mutant was obtained under anaerobic conditions in the presence of dithiothreitol (DTT). Unexpectedly, the trxB1 mutant was viable without DTT and under aerated static conditions, thus disproving the essentiality of this system. Aerobic growth of the trxB1 mutant did not require glutathione, also ruling out the need for this redox maintenance system. Proteomic analyses showed that known oxidative stress defense proteins are induced in the trxB1 mutant. Two additional effects of trxB1 were not previously reported in other bacteria: (i) induction of proteins involved in fatty acid or menaquinone biosynthesis, indicating that membrane synthesis is part of the cellular response to a redox imbalance, and (ii) alteration of the isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapB). We determined that the two GapB isoforms in L. lactis differed by the oxidation state of catalytic-site cysteine C152. Unexpectedly, a decrease specific to the oxidized, inactive form was observed in the trxB1 mutant, possibly because of proteolysis of oxidized GapB. This study showed that thioredoxin reductase is not essential in L. lactis and that its inactivation triggers induction of several mechanisms acting at the membrane and metabolic levels. The existence of a novel redox function that compensates for trxB1 deficiency is suggested.
Organic thiols are toxic to eukaryotic cells. Treatment of cells with thiols activates exporession of grp78, but it is not known if, like other forms of strees, there is a battery of stress response genes that are induced by thiols, In LLC-PK1 renal epithelial cells, mRNAs for both grp78 and gadd153 were induced by thiols with similar time, concentration and structure-activity dependence. Dithiothreitol (DTT) was the most potent reductant and inducer of gene expression among the thiols tested. Nuclear run-on assays demonstrated that DTT activated both grp78 and gadd153 genes transcriptionally. A hamster gadd153 promoter construct which contains enhancer elements necessary for gadd153 activation was stably integrated into the LLC-PK1 cell genome and was activated by DTT. Although auto-oxidation of thiols can generate active oxygen species, trtanscriptional activation of the gadd153 promoter was not due to formation of hydrogen peroxide or superoxide since neither catalase nor superoxide dismutase prevented activation of the inhibition of dome formation and protein synthesis, two toxic effects of DTT in LLC-PK1 cells. Thus, both grp78 and gadd153 are members of a gene battery which is responsive to reductive stress. There appears to be considerable, but not complete, overlap between the upstream signaling pathways for activation of both genes.
TGF-β plays an important role in the genesis and progression of pulmonary fibrosis. We sought to determine the role of mononuclear phagocytes in the activation of TGF-β and found that freshly isolated peripheral blood monocytes spontaneously released TGF-β. Stimulating these monocytes with GM-CSF or LPS, but not MCSF, augmented the activation of TGF-β. In human monocytes, the free thiol compounds DTT and NAC decreased the activity of TGF-β, without affecting TGF-β mRNA transcription. Both NAC and DTT lessened the biological activity of recombinant active TGF-β in a cell-free system. We found that NAC and DTT reduced dimeric active TGF-β from a 25 kDa protein to 12.5 kDa inactive monomer. This conversion was reversed using the oxidizing agent diamide. Diamide also restored biological activity to NAC or DTT-treated TGF-β. Reduction of TGF-β to monomers could competitively inhibit active dimeric TGF-β and block intracellular signaling events. Our observations suggest that modulation of the oxidative state of TGF-β may be a novel therapeutic approach for patients with pulmonary fibrosis.
In the title compound, C16H13Cl2N3O2S, the dihedral angle between the aromatic rings is 6.62 (2)° and the C=N—N=C torsion angle is 176.2 (4)°. The thiazine ring shows an envelope conformation, with the S atom displaced by 0.633 (6) Å from the mean plane of the other five atoms (r.m.s. deviation = 0.037 Å). The Cl atom is an an axial conformation and is displaced by 2.015 (6) Å from the thiazine ring plane. In the crystal, molecules are linked by C—H⋯O interactions, generating a three-dimensional network. Very weak aromatic π–π stacking interactions [centroid–centroid separations = 3.928 (2) Å] are also observed.
Liquefaction and homogenization have been recommended to ensure accurate, representative sputum cultures. We evaluated dithiothreitol (DTT) as mucolytic agent for culturing sputum samples obtained from 79 cystic fibrosis (CF) patients. Liquefaction with DTT was not superior to direct plating of specimens for routine qualitative cultures. Unliquefied sputum cultures failed to direct 3 of 47 Pseudomonas aeruginosa isolates; DTT-treated specimens missed 5 of 13 Candida albicans isolates. Neither treated nor untreated sputum cultures were completely successful in detecting Staphylococcus aureus or Enterobacteriaceae. Since Haemophilus influenzae was recovered from only two qualitative cultures, we could not evaluate the effect of DTT on the receovery of this organism. However, 27 of 29 strains of H. influenzae were inhibited by concentrations of DTT near the recommended final working concentration of 50 micrograms/ml, suggesting that liquefaction might impair isolation of this organism. Liquefaction with DTT permitted quantitative cultures of CF sputum. The predominant pathogen in our CF population was P. aeruginosa; 37 of 43 (86%) patients were colonized with this organism. Median densities of rough and mucoid strains were 3.2 x 10(7) and 4.3 x 10(7) colony-forming units per ml, respectively. Previous oral antistaphylococcal therapy may have accounted for the observed low density of S. aureus (mean density, 3.5 x 10(3) colony-forming units per ml). We conclude that DTT treatment does not improve recovery of organisms from qualitative cultures but does facilitate quantitative studies of S. aureus and P. aeruginosa in CF sputum.
Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers.