Thiol Modification Research Articles

A simple route to transform normal hydrophilic cloth into a superhydrophobic–superhydrophilic hybrid surface

By utilizing the selective modification of n-octadecyl thiol for the Fe and Co nanoparticles, we fabricated superhydrophobic–superhydrophilic hybrid fabrics by a simple in situ growth process.

Cyclic Voltammetric Studies on the Role of Electrode, Electrode Surface Modification and Electrolyte Solution of an Electrochemical Cell

The present study has been aimed to understand the role of electrode material, its surface modification by organic molecules, solvent and analyte in an electrochemical cell on cyclic voltammograms. To study these effects, the different electrochemical cells were constituted using different electrode material, different solvents and thiol modification of electrode surface to measure the cyclic voltammograms. The present measurements clearly show that these parameters are significantly important ones in dictating the nature of the observed voltammograms. The effect of scan rate on cyclic voltammograms has been observed with various scan rates. A plot of the Randles-Sevcik equation yield a straight line, the slope is used to determine the diffusion coefficient. Since the cyclic voltammetry is an important technique in the characterization and in the development of one of the important energy storage devices that are known as supercapacitors. The present investigations clearly help to understand the internal mechanisms of energy storage in these devices so that the better devices can be developed.

Zebrafish heart development is regulated via glutaredoxin 2 dependent migration and survival of neural crest cells

Glutaredoxin 2 is a vertebrate specific oxidoreductase of the thioredoxin family of proteins modulating the intracellular thiol pool. Thereby, glutaredoxin 2 is important for specific redox signaling and regulates embryonic development of brain and vasculature via reversible oxidative posttranslational thiol modifications. Here, we describe that glutaredoxin 2 is also required for successful heart formation. Knock-down of glutaredoxin 2 in zebrafish embryos inhibits the invasion of cardiac neural crest cells into the primary heart field. This leads to impaired heart looping and subsequent obstructed blood flow. Glutaredoxin 2 specificity of the observed phenotype was confirmed by rescue experiments. Active site variants of glutaredoxin 2 revealed that the (de)-glutathionylation activity is required for proper heart formation. Our data suggest that actin might be one target during glutaredoxin 2 regulated cardiac neural crest cell migration and embryonic heart development. In summary, this work represents further evidence for the general importance of redox signaling in embryonic development and highlights additionally the importance of glutaredoxin 2 during embryogenesis.

Functional DNA Nanostructures for Theranostic Applications

There has been tremendous interest in constructing nanostructures by exploiting the unparalleled ability of DNA molecules in self-assembly. We have seen the appearance of many fantastic, "art-like" DNA nanostructures in one, two, or three dimensions during the last two decades. More recently, much attention has been directed to the use of these elegant nanoobjects for applications in a wide range of areas. Among them, diagnosis and therapy (i.e., theranostics) are of particular interest given the biological nature of DNA. One of the major barricades for the biosensor design lies in the restricted target accessibility at the solid-water interface. DNA nanotechnology provides a convenient approach to well control the biomolecule-confined surface to increase the ability of molecular recognition at the biosensing interface. For example, tetrahedral DNA nanostructures with thiol modifications can be self-assembled at the gold surface with high reproducibility. Since DNA tetrahedra are highly rigid and well-defined structures with atomic precision and versatile functionality, they provide scaffolds for anchoring of a variety of biomolecular probes (DNA, aptamers, peptides, and proteins) for biosensing. Significantly, this DNA nanostructure-based biosensing platform greatly increases target accessibility and improves the sensitivity for various types of molecular targets (DNA, RNA, proteins, and small molecules) by several orders of magnitude. In an alternative approach, DNA nanostructures provide a framework for the development of dynamic nanosensors that can function inside the cell. DNA tetrahedra are found to be facilely cell permeable and can sense and image specific molecules in cells. More importantly, these DNA nanostructures can be efficient drug delivery nanocarriers. Since they are DNA molecules by themselves, they have shown excellent cellular biocompatibility with minimal cytotoxicity. As an example, DNA tetrahedra tailored with CpG oligonucleotide drugs have shown greatly improved immunostimulatory effects that makes them a highly promising nanomedicine. By taking them together, we believe these functionalized DNA nanostructures can be a type of intelligent theranostic nanodevice for simultaneous sensing, diagnosis, and therapy inside the cell.

Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications

Reversible modifications of cysteine thiols have a key role in redox signaling and regulation. A number of reversible redox modifications, including disulfide formation, S-nitrosylation (SNO) and S-glutathionylation (SSG), have been recognized for their significance in various physiological and pathological processes. Here we describe a procedure for the enrichment of peptides containing reversible cysteine modifications. Starting with tissue or cell lysate samples, all of the unmodified free thiols are blocked using N-ethylmaleimide (NEM). This is followed by the selective reduction of those cysteines bearing the reversible modification(s) of interest. The reduction is achieved by using different reducing reagents that react specifically with each type of cysteine modification (e.g., ascorbate for SNO). This protocol serves as a general approach for enrichment of thiol-containing proteins or peptides derived from reversibly modified proteins. The approach uses a commercially available thiol-affinity resin (thiopropyl Sepharose 6B) to directly capture free thiol-containing proteins through a disulfide exchange reaction, followed by on-resin protein digestion and multiplexed isobaric labeling to facilitate liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitative site-specific analysis of cysteine-based reversible modifications. The overall approach requires a simpler workflow with increased specificity compared with the commonly used biotinylation-based assays. The procedure for selective enrichment and analyses of SNO and the level of total reversible cysteine modifications (or total oxidation) is presented to demonstrate the utility of this general strategy. The entire protocol requires ∼3 d for sample processing with an additional day for LC-MS/MS and data analysis.

Thermal Aggregation Properties of Nanoparticles Modified with Temperature Sensitive Copolymers

In this paper, we describe the use of a temperature responsive polymer to reversibly assemble gold nanoparticles of various sizes. Temperature responsive, low critical solution temperature (LCST) pNIPAAm-co-pAAm polymers, with transition temperatures (T(C)) of 51 and 65 °C, were synthesized with a thiol modification, and grafted to the surface of 11 and 51 nm gold nanoparticles (AuNPs). The thermal-responsive behavior of the polymer allowed for the reversible aggregation of the nanoparticles, where at T < T(C) the polymers were hydrophilic and extended between particles. In contrast, at T > T(C), the polymer shell undergoes a hydrophilic to hydrophobic phase transition and collapses, decreasing interparticle distances between particles, allowing aggregation to occur. The AuNP morphology and polymer conjugation were probed by TEM, FTIR, and (1)H NMR. The thermal response was probed by UV-vis and DLS. The structure of the assembled aggregates at T > T(C) was studied via in situ small-angle X-ray scattering, which revealed interparticle distances defined by polymer conformation.

Molecular mechanism and physiological role of active–deactive transition of mitochondrial complex I

The unique feature of mitochondrial complex I is the so-called A/D transition (active–deactive transition). The A-form catalyses rapid oxidation of NADH by ubiquinone (k ~104 min−1) and spontaneously converts into the D-form if the enzyme is idle at physiological temperatures. Such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. The D-form can undergo reactivation given both NADH and ubiquinone availability during slow (k ~1–10 min−1) catalytic turnover(s). We examined known conformational differences between the two forms and suggested a mechanism exerting A/D transition of the enzyme. In addition, we discuss the physiological role of maintaining the enzyme in the D-form during the ischaemic period. Accumulation of the D-form of the enzyme would prevent reverse electron transfer from ubiquinol to FMN which could lead to superoxide anion generation. Deactivation would also decrease the initial burst of respiration after oxygen reintroduction. Therefore the A/D transition could be an intrinsic protective mechanism for lessening oxidative damage during the early phase of reoxygenation. Exposure of Cys39 of mitochondrially encoded subunit ND3 makes the D-form susceptible for modification by reactive oxygen species and nitric oxide metabolites which arrests the reactivation of the D-form and inhibits the enzyme. The nature of thiol modification defines deactivation reversibility, the reactivation timescale, the status of mitochondrial bioenergetics and therefore the degree of recovery of the ischaemic tissues after reoxygenation.

S-Glutathionylation of Ion Channels: Insights into the Regulation of Channel Functions, Thiol Modification Crosstalk, and Mechanosensing

Ion channels control membrane potential, cellular excitability, and Ca(++) signaling, all of which play essential roles in cellular functions. The regulation of ion channels enables cells to respond to changing environments, and post-translational modification (PTM) is one major regulation mechanism. Many PTMs (e.g., S-glutathionylation, S-nitrosylation, S-palmitoylation, S-sulfhydration, etc.) targeting the thiol group of cysteine residues have emerged to be essential for ion channels regulation under physiological and pathological conditions. Under oxidative stress, S-glutathionylation could be a critical PTM that regulates many molecules. In this review, we discuss S-glutathionylation-mediated structural and functional changes of ion channels. Criteria for testing S-glutathionylation, methods and reagents used in ion channel S-glutathionylation studies, and thiol modification crosstalk, are also covered. Mechanotransduction, and S-glutathionylation of the mechanosensitive KATP channel, are discussed. Further investigation of the ion channel S-glutathionylation, especially the physiological significance of S-glutathionylation and thiol modification crosstalk, could lead to a better understanding of the thiol modifications in general and the ramifications of such modifications on cellular functions and related diseases.

Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

BackgroundCucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology.FindingsCucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation.ConclusionsThe implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution.

Using Temperature-Sensitive Smart Polymers to Regulate DNA-Mediated Nanoassembly and Encoded Nanocarrier Drug Release

In this paper we describe the use of a temperature-responsive polymer to regulate DNA interactions in both a DNA-mediated assembly system and a DNA-encoded drug delivery system. A thermoresponsive pNIPAAm-co-pAAm polymer, with a transition temperature (TC) of 51 °C, was synthesized with thiol modification and grafted onto gold nanoparticles (Au NPs) also containing single-stranded oligonucleotides (ssDNA). The thermoresponsive behavior of the polymer regulated the accessibility of the sequence-specific hybridization between complementary DNA-functionalized Au NPs. At T < TC, the polymer was hydrophilic and extended, blocking interaction between the complementary sequences at the periphery of the hydrodynamic diameter. In contrast, at T > TC, the polymer shell undergoes a hydrophilic to -phobic phase transition and collapses, shrinking below the outer ssDNA, allowing for the sequence-specific hybridization to occur. The potential application of this dynamic interface for drug delivery is shown, in which the chemotherapy drug doxorubicin (DOX) is bound to double-stranded DNA (dsDNA)-functionalized Au NPs whose sequences are known to be high-affinity intercalation points for it. The presence of the polymer capping is shown to decrease drug release kinetics and equilibrium at T < TC, but increase release at T > TC, thus improving the cytotoxicity of the encoded nanocarrier design.

Mechanics of pH-Responsive Hydrogel Capsules

While soft hydrogel nano- and microstructures hold great potential for therapeutic treatments and in vivo applications, their nanomechanical characterization remains a challenge. In this paper, soft, single-component, supported hydrogel films were fabricated using pendant-thiol-modified poly(methacrylic acid) (PMASH). The influence of hydrogel architecture on deformation properties was studied by fabricating films on particle supports and producing free-standing capsules. The influence of the degree of thiol-based cross-linking on the mechanical properties of the soft hydrogel systems (core-shell and capsules) was studied using a colloidal-probe (CP) AFM technique. It was found that film mechanical properties, stability, and capsule swelling could be finely tuned by controlling the extent of poly(methacrylic acid) thiol modification. Furthermore, switching the pH from 7.4 to 4.0 led to film densification due to increased hydrogen bonding. Hydrogel capsule systems were found to have stiffness values ranging from 0.9 to 16.9 mN m(-1) over a thiol modification range of 5 to 20 mol %. These values are significantly greater than those for previously reported PMASH planar films of 0.7-5.7 mN m(-1) over the same thiol modification range (Best et al., Soft Matter 2013, 9, 4580-4584). Films on particle substrates had comparable mechanical properties to planar films, demonstrating that while substrate geometry has a negligible effect, membrane and tension effects may play an important role in capsule force resistance. Further, when transitioning from solid-supported films to free-standing capsules, simple predictions of shell stiffness based on modulus changes found for supported films are not valid. Rather, additional effects like diameter increases (geometrical changes) as well as tension buildup need to be taken into account. These results are important for research related to the characterization of soft hydrogel materials and control over their mechanical properties.

Cilia-Mimetic Hairy Surfaces Based on End-Immobilized Nanocellulose Colloidal Rods

We show a simple method toward nanoscale cilia-like structures, i.e., functional hairy surfaces, upon topochemically functionalizing nanorods of cellulose nanocrystals (CNCs) with thiol end groups (CNC-SHs), which leads to their immobilization onto a gold surface from one end, still allowing their orientational mobility. CNCs having a lateral dimension of 3-5 nm and length of 50-500 nm incorporate the native crystalline structure with hydrogen-bonded cellulose chains in the parallel configuration. This facilitates asymmetric, selective chemical modification of the reducing ends through reductive amination. Successful thiol functionalization is demonstrated using cryo transmission electron microscopy based on selective attachment of silver nanoparticles to the CNC-SH ends to form Janus-like colloidal rod-sphere adducts. The extent of thiol modification of CNC-SHs is quantified using X-ray photoelectron spectroscopy. The promoted binding of CNC-SHs on gold surfaces is shown by atomic force microscopy and quartz crystal microbalance, where the high dissipation suggests pronounced orientational mobility due to flexible joints at one rod end onto the surfaces. That the joints are flexible is also shown by the bending and realignment of the CNC-SH rods using a receding triple-phase evaporation front of a drying drop of water. The ability of the hairy surface to size-selectively resist particle binding was also investigated. As the CNCs are piezoelectric and allow magnetic functionalization by nanoparticles, we foresee a general platform for nanosized artificial cilia for fluid manipulation and controlled adsorption/desorption.

Redox Proteomics Uncovers Peroxynitrite-sensitive Proteins That Help Escherichia coli to Overcome Nitrosative Stress

Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.

Polysulfides Link H2S to Protein Thiol Oxidation

Hydrogen sulfide (H2S) is suggested to act as a gaseous signaling molecule in a variety of physiological processes. Its molecular mechanism of action was proposed to involve protein S-sulfhydration, that is, conversion of cysteinyl thiolates (Cys-S(-)) to persulfides (Cys-S-S(-)). A central and unresolved question is how H2S-that is, a molecule with sulfur in its lowest possible oxidation state (-2)-can lead to oxidative thiol modifications. Using the lipid phosphatase PTEN as a model protein, we find that the "H2S donor" sodium hydrosulfide (NaHS) leads to very rapid reversible oxidation of the enzyme in vitro. We identify polysulfides formed in NaHS solutions as the oxidizing species, and present evidence that sulfane sulfur is added to the active site cysteine. Polysulfide-mediated oxidation of PTEN was induced by all "H2S donors" tested, including sodium sulfide (Na2S), gaseous H2S, and morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137). Moreover, we show that polysulfides formed in H2S solutions readily modify PTEN inside intact cells. Our results shed light on the previously unresolved question of how H2S leads to protein thiol oxidation, and suggest that polysulfides formed in solutions of H2S mediate this process. This study suggests that the effects that have been attributed to H2S in previous reports may in fact have been mediated by polysulfides. It also supports the notion that sulfane sulfur rather than sulfide is the actual in vivo agent of H2S signaling.

Genistein attenuates ischemic oxidative damage and behavioral deficits via eNOS/Nrf2/HO-1 signaling

Global cerebral ischemia, such as occurs following cardiac arrest, can lead to oxidative stress, hippocampal neuronal cell death, and cognitive defects. The current study examined the potential beneficial effect and underlying mechanisms of post-treatment with the naturally occurring isoflavonic phytoestrogen, genistein, which has been implicated to attenuate oxidative stress. Genistein (1 mg kg(-1)) was administered i.v. 5 min after reperfusion in rats subjected to four-vessel global cerebral ischemia (GCI). The results revealed that genistein exerted significant neuroprotection of hippocampal CA1 neurons following GCI, as evidenced by an increase in NeuN-positive neurons and the decrease in TUNEL-positive neurons. Furthermore, genistein treatment also resulted in significantly improved spatial learning and memory as compared to vehicle control animals. The beneficial effects of genistein appear to be mediated by an increase of phosphorylation/activation of eNOS, with subsequent activation of the antioxidant/detoxification Nrf2/Keap1 transcription system. Along these lines, genistein increased keap1 S-nitrosylation, with a corresponding nuclear accumulation and enhanced DNA binding activity of Nrf2. Genistein also enhanced levels of the Nrf2 downstream antioxidant protein, heme oxygenase (HO)-1, as compared to vehicle control groups. In accordance with its induction of Nrf2 activation, genistein exerted a robust attenuation of oxidative DNA damage and lipid peroxidative damage in hippocampal CA1 neurons after GCI, as measured by immunofluorescence staining of the oxidative stress markers, 8-hydroxy-2-deoxyguanosine (8-OHdG) and 4-Hydroxynonenal (4-HNE). Interestingly, the aforementioned effects of genistein were abolished by pretreatment with L-NAME, an inhibitor of eNOS activation. In conclusion, the results of the study demonstrate that low dose genistein can exert significant antioxidant, neuroprotective, and cognitive-enhancing effects in the hippocampal CA1 region following GCI. Mechanistically, the beneficial effects of genistein appear to be mediated by enhanced eNOS phosphorylation/activation and nitric oxide (NO)-mediated thiol modification of Keap1, with subsequent upregulation of the Nrf2/HO-1 antioxidative signaling pathway and a resultant attenuation of oxidative stress.

Protein expression profiling of nuclear membrane protein reveals potential biomarker of human hepatocellular carcinoma

BackgroundComplex molecular events lead to development and progression of liver cirrhosis to HCC. Differentially expressed nuclear membrane associated proteins are responsible for the functional and structural alteration during the progression from cirrhosis to carcinoma. Although alterations/ post translational modifications in protein expression have been extensively quantified, complementary analysis of nuclear membrane proteome changes have been limited. Deciphering the molecular mechanism that differentiate between normal and disease state may lead to identification of biomarkers for carcinoma.ResultsMany proteins displayed differential expression when nuclear membrane proteome of hepatocellular carcinoma (HCC), fibrotic liver, and HepG2 cell line were assessed using 2-DE and ESI-Q-TOF MS/MS. From the down regulated set in HCC, we have identified for the first time a 15 KDa cytochrome b5A (CYB5A), ATP synthase subunit delta (ATPD) and Hemoglobin subunit beta (HBB) with 11, 5 and 22 peptide matches respectively. Furthermore, nitrosylation studies with S-nitrosocysteine followed by immunoblotting with anti SNO-cysteine demonstrated a novel and biologically relevant post translational modification of thiols of CYB5A in HCC specimens only. Immunofluorescence images demonstrated increased protein S-nitrosylation signals in the tumor cells and fibrotic region of HCC tissues. The two other nuclear membrane proteins which were only found to be nitrosylated in case of HCC were up regulated ATP synthase subunit beta (ATPB) and down regulated HBB. The decrease in expression of CYB5A in HCC suggests their possible role in disease progression. Further insight of the functional association of the identified proteins was obtained through KEGG/ REACTOME pathway analysis databases. String 8.3 interaction network shows strong interactions with proteins at high confidence score, which is helpful in characterization of functional abnormalities that may be a causative factor of liver pathology.ConclusionThese findings may have broader implications for understanding the mechanism of development of carcinoma. However, large scale studies will be required for further verification of their critical role in development and progression of HCC.

Structural Characteristics of the Redox-sensing Coiled Coil in the Voltage-gated H+ Channel

Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H(+) (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys → Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome.

Isoferulic Acid, a New Anti-Glycation Agent, Inhibits Fructose- and Glucose-Mediated Protein Glycation in Vitro

The inhibitory activity of isoferulic acid (IFA) on fructose- and glucose-mediated protein glycation and oxidation of bovine serum albumin (BSA) was investigated. Our data showed that IFA (1.25–5 mM) inhibited the formation of fluorescent advanced glycation end products (AGEs) and non-fluorescent AGE [Nε-(carboxymethyl) lysine: CML], as well as the level of fructosamine. IFA also prevented protein oxidation of BSA indicated by decreasing protein carbonyl formation and protein thiol modification. Furthermore, IFA suppressed the formation of β-cross amyloid structures of BSA. Therefore, IFA might be a new promising anti-glycation agent for the prevention of diabetic complications via inhibition of AGEs formation and oxidation-dependent protein damage.

P38: Mechanisms for cross-membrane vasodilatory signaling of nitrosothiols in isolated sheep femoral arteries

Nitrosothiols (SNOs) are proposed to play an important role in nitric oxide (NO) mediated vasodilation. Vasoactivity of SNOs rely, at least in part, on transfer of NO equivalent from extracellular SNOs into smooth muscle cells. There are several possible pathways for this cross-membrane vasodilatory signaling. First, SNOs may release free NO (NO ) outside the cell membrane followed by diffusion of the NO into cell to activate guanylyl cyclase. Second, SNOs may be taken up into cell via l -type amino acid transporter (LAT) before releasing NO within cell. A third possibility is that SNOs may transnitrosate thiol groups on cell membrane to initiate trans-membrane signaling events. These experiments test for the role of these three possibilities by using selective blockers of each pathway, and by comparison of vasodilating effects of SNOs which have distinct stereo conformation and membrane permeability. Endothelium-denuded sheep femoral arterial segments were mounted on a wire myograph, pre-contracted with phenylephrine (10 μmol/L), and then subjected to dose–responses to various SNOs. To test the extent to which SNO-mediated dilation occurs by NO activation of guanylyl cyclase, experiments were carried out in the presence of the competitive guanylyl cyclase inhibitor ODQ (10 μmol/L). To test whether SNO-mediated dilation involves the release of NO outside the cell, experiments were performed in the presence of CPTIO (200 μmol/L), a membrane impermeable NO scavenger. To test for a role for the stereo-selective LAT, experiments were performed with S -nitroso- l -cysteine ( l -cysNO) which is a substrate of LAT, compared to its stereoisomer S -nitroso- d -cysteine ( d -cysNO). In addition, we measured responses to the membrane impermeable low and high molecular weight SNOs, S -nitroso-glutathione (GSNO) and S -nitroso-albumin (AlbSNO). Experiments were also performed in the presence of threonine (10 mmol/L) and BCH (10 mmol/L), inhibitors of the LAT. To test for the possibility of transnitrosation of membrane thiols, experiments were carried out after incubation of vessels with impermeable thiol modifier HMBA (10 μmol/L; extensively washed out before application of SNOs to artery). The vasodilation of l -cysNO was significantly attenuated by competitive guanylyl cyclase inhibitor ODQ and partially attenuated by impermeable NO scavenger CPTIO. Surprisingly, vasodilation was significantly potentiated by the LAT inhibitors threonine and BCH. Vasodilation was not affected by blocking extracellular thiols with HMBA. The vasoactivities of d -cysNO, GSNO and AlbSNO were similar ( p > 0.05 for EC 50 s) to that of l -cysNO. In addition, the effects of ODQ, CPTIO, threonine, BCH and HMBA on d -cysNO were all similar ( p > 0.05 for EC 50 s) to that on l -cysNO. Our results suggest that the SNO-derived vasodilating NO equivalent is not NO released outside the cell membrane. Besides, uptake of SNO as a whole is not a prerequisite for its vasodilation, but weakens vasodilation instead. Further more, membrane thiol was not involved in the cross-membrane vasodilatory signaling of SNOs. Nothing to disclose.

Structural Studies of RGS Inhibitors

Regulators of G‐protein signaling (RGS) proteins regulate G‐protein coupled receptor (GPCR) signal transduction cascades by acting as GTPase accelerating proteins upon binding activated Gα subunits. RGS proteins express in cell‐type specific manners, regulate specific GPCRs, and are overexpressed in pathologies from cancer to Parkinson's disease. As such, they are attractive drug targets. Small‐molecule RGS4 inhibitor screens have yielded a number of covalent thiol modifiers that act allosterically at Cys148, far from the RGS/Gα interface. Thus far, the structural basis of RGS4 inhibition is unknown, so in the current study we investigate the structural consequence of RGS4's covalent modification that leads to inhibition of Gα binding. We purified recombinant RGS4 from Escherichia coli and obtained nuclear magnetic resonance spectra for free and modified RGS4. Overlaid 15N‐1H HSQC spectra show significant changes in RGS4 structure upon small molecule modification. Data from circular dichroism, dynamic light scattering, and other techniques provide further evidence that the pocket may be amenable for rational drug design. Project funded by 5T32GM067795–08 and pre‐doctoral fellowship from AFPE.