N-ethylmaleimide Research Articles

Enhancing the peroxidase activity and decreasing the protease activity of ficin with rational modification and its application to one-step colorimetric detection of glucose.

Ficin has dual enzyme activity, i.e., protease and peroxidase-like activity. In some respects, its application is limited by the protease activity of ficin. Herein, we used tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to break the three pairs of disulfide bonds of ficin, and then blocked the free thiol groups with N-ethylmaleimide (NEM) to synthesize ficin-TN. The results showed that ficin-TN had increased peroxidase-like activity and reduced protease activity. According to this phenomenon, we have exploited a colorimetric method with high sensitivity and selectivity for the one-step detection of glucose. Comparing with ficin, ficin-TN has wider detection range (0.1-300μM) and lower detection limit (88nM), and our method is simpler and more timesaving than other two-step methods. Furthermore, the actual appliances of ficin-TN for glucose detection in human serum have been illustrated with satisfied result, suggesting that its promising utilization in various fields.

Synthesis of Well-Defined Alternating Copolymer Composed of Ethylmaleimide and Hydroxy-Functionalized Vinyl Ether by RAFT Polymerization and Their Thermoresponsive Properties.

Here we report the controlled synthesis of alternating copolymers by reversible addition-fragmentation chain transfer (RAFT) polymerization of hydroxy-functionalized vinyl ether (DEGV) and ethylmaleimide (EtMI) using dithiocarbonate derivative (CPDB) as the RAFT reagent. The resulting alternating copolymer poly[ethylmaleimide-alt-(diethylene glycol mono vinyl ether)] (poly(MalMI-alt-DEGV)) had a relatively narrow molecular weight distribution (Mw/Mn < 1.4). These polymers are fully soluble in cold water (5 °C) and an aqueous solution of poly(MalMI-alt-DEGV) became turbid upon heating (using an incident wavelength of 600 nm and 1.0 mg mL−1 (0.1 wt %) polymer concentration), indicating phase separation above the cloud point temperature (Tcp). The Tcp of the polymer solution ranged from 15–35 °C, depending on the molecular weight and molecular weight distribution of the polymer.

Optimized Protocol for the In Situ Derivatization of Glutathione with N-Ethylmaleimide in Cultured Cells and the Simultaneous Determination of Glutathione/Glutathione Disulfide Ratio by HPLC-UV-QTOF-MS.

Glutathione (GSH) and glutathione disulfide (GSSG) are commonly used to assess the oxidative status of a biological system. Various protocols are available for the analysis of GSH and GSSG in biomedical specimens. In this study, we present an optimized protocol for the in situ derivatization of GSH with N-ethylmaleimide (NEM) to prevent GSH autooxidation, and thus to preserve the GSH/GSSG ratio during sample preparation. The protocol comprises the incubation of cells in NEM containing phosphate buffered saline (PBS), followed by metabolite extraction with 80% methanol. Further, to preserve the use of QTOF-MS, which may lack the linear dynamic range required for the simultaneous quantification of GSH and GSSG in non-targeted metabolomics, we combined liquid chromatographic separation with the online monitoring of UV absorbance of GS-NEM at 210 nm and the detection of GSSG and its corresponding stable isotope-labeled internal standard by QTOF-MS operated with a 10 Da Q1 window. The limit of detection (LOD) for GS-NEM was 7.81 µM and the linear range extended from 15.63 µM to 1000 µM with a squared correlation coefficient R2 of 0.9997. The LOD for GSSG was 0.001 µM, and the lower limit of quantification (LLOQ) was 0.01 µM, with the linear (R2 = 0.9994) range extending up to 10 µM. The method showed high repeatability with intra-run and inter-run coefficients of variation of 3.48% and 2.51% for GS-NEM, and 3.11% and 3.66% for GSSG, respectively. Mean recoveries of three different spike-in levels (low, medium, high) of GSSG and GS-NEM were above 92%. Finally, the method was applied to the determination of changes in the GSH/GSSG ratio either in response to oxidative stress in cells lacking one or both monocarboxylate transporters MCT1 and MCT4, or in adaptation to the NADPH (nicotinamide adenine dinucleotide phosphate) consuming production of D-2-hydroxyglutarate in cells carrying mutations in the isocitrate dehydrogenase genes IDH1 and IDH2.

Ag+ Ion Binding to Human Metallothionein-2A Is Cooperative and Domain Specific.

Metallothioneins (MTs) constitute a family of cysteine-rich proteins that play key biological roles for a wide range of metal ions, but unlike many other metalloproteins, the structures of apo- and partially metalated MTs are not well understood. Here, we combine nano-electrospray ionization-mass spectrometry (ESI-MS) and nano-ESI-ion mobility (IM)-MS with collision-induced unfolding (CIU), chemical labeling using N-ethylmaleimide (NEM), and both bottom-up and top-down proteomics in an effort to better understand the metal binding sites of the partially metalated forms of human MT-2A, viz., Ag4-MT. The results for Ag4-MT are then compared to similar results obtained for Cd4-MT. The results show that Ag4-MT is a cooperative product, and data from top-down and bottom-up proteomics mass spectrometry analysis combined with NEM labeling revealed that all four Ag+ ions of Ag4-MT are bound to the β-domain. The binding sites are identified as Cys13, Cys15, Cys19, Cys21, Cys24, and Cys26. While both Ag+ and Cd2+ react with MT to yield cooperative products, i.e., Ag4-MT and Cd4-MT, these products are very different; Ag+ ions of Ag4-MT are located in the β-domain, whereas Cd2+ ions of Cd4-MT are located in the α-domain. Ag6-MT has been reported to be fully metalated in the β-domain, but our data suggest the two additional Ag+ ions are more weakly bound than are the other four. Higher order Agi-MT complexes (i = 7-17) are formed in solutions that contain excess Ag+ ions, and these are assumed to be bound to the α-domain or shared between the two domains. Interestingly, the excess Ag+ ions are displaced upon addition of NEM to this solution to yield predominantly Ag4NEM14-MT. Results from CIU suggest that Agi-MT complexes are structurally more ordered and that the energy required to unfold these complexes increases as the number of coordinated Ag+ increases.

Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1.

The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.

Absolute Modification of Highly Reactive Cysteines via N‐ethylmaleimide Evokes Full, Prolonged TRPA1 Activation

A subgroup of sensory nerve fibers called nociceptors is stimulated by noxious stimuli and evokes defensive responses in organ systems. Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, non‐selective, cationic channel that is expressed in sensory neurons that initiate afferent signaling in nociceptive fibers when activated. Activation of TRPA1 initiates nociceptive signaling to the central nervous system and elicits defensive behaviors and reflexes in organ systems and throughout the body. TRPA1 is activated by environmental pollutants, exogenous food chemicals and endogenous irritants that are created during inflammation and oxidative stress. TRPA1 agonists are electrophilic compounds that covalently modify highly reactive cysteines on the intracellular side of the channel. Our lab has identified four highly reactive cysteines (C273, C621, C665, C1085) that rapidly bind to electrophiles, with C621 having a greater reactivity than the other three cysteines. It is known that electrophilic binding of cysteines contributes to TRPA1 activation; however, the underlying mechanism of activation is poorly understood. In this study, we utilize cell‐attached single channel patch‐clamp to observe stochastic gating events of human TRPA1 (hTRPA1) channels that is transiently expressed in HEK293 cells to better understand the mechanism that leads to TRPA1 activation. Recordings of stochastic TRPA1 gating events during N‐ethylmaleimide (NEM) treatment reveal two different activation profiles: (1) a weak activation (low open probability [Po]) that persists over time; (2) a weak activation followed by a rapid, spontaneous increase to full activation (near 100% Po). Mean open time histogram analysis shows that the full TRPA1 activation profile has longer event open time compared to weak TRPA1 activation, which has mean open times that are comparable to open times of random events during baseline conditions. Also, the mean maximum open times during full TRPA1 activation is 75 times longer than weak TRPA1 activation mean maximum open times. Our results suggest that differences in the full or weak TRPA1 activation profiles are due to a difference in how the cysteines are modified via NEM. We hypothesize that full activation is the result of absolute C621 modification (each C621 residue on the four TRPA1 subunits) that fully opens the channel, whereas weak activation is the result of individual C621 modification events (not all four C621 residues) that do not have an additive effect on TRPA1 channel activation.

Coexistence of D3 R typical and atypical signaling in striatonigral neurons during dopaminergic denervation. Correlation with D3 nf expression changes.

Dopamine D3 R are widely expressed in basal ganglia where interact with D1 R. D3 R potentiate cAMP accumulation and GABA release stimulated by D1 R in striatonigral neurons through "atypical" signaling. During dopaminergic denervation, D3 R signaling changes to a "typical" in which antagonizes the effects of D1 R, the mechanisms of this switching are unknown. D3 nf splice variant regulates membrane anchorage and function of D3 R and decreases in denervation; thus, it is possible that D3 R signaling switching correlates with changes in D3 nf expression and increases of membranal D3 R that mask D3 R atypical effects. We performed experiments in unilaterally 6-hydroxydopamine lesioned rats and found a decrease in mRNA and protein of D3 nf, but not of D3 R in the denervated striatum. Proximity ligation assay showed that D3 R-D3 nf interaction decreased after denervation, whereas binding revealed an increased Bmax in D3 R. The new D3 R antagonized cAMP accumulation and GABA release stimulated by D1 R; however, in the presence of N-Ethylmaleimide (NEM), to block Gi protein signaling, activation of D3 R produced its atypical signaling stimulating D1 R effects. Finally, we investigated if the typical and atypical effects of D3 R modulating GABA release are capable of influencing motor behavior. Injections of D3 R agonist into denervated nigra decreased D1 R agonist-induced turning behavior but potentiated it in the presence of NEM. Our data indicate the coexistence of D3 R typical and atypical signaling in striatonigral neurons during denervation that correlated with changes in the ratio of expression of D3 nf and D3 R isoforms. The coexistence of both atypical and typical signaling during denervation influences motor behavior.

Diallyl Trisulfide Inhibits Platelet Aggregation through the Modification of Sulfhydryl Groups.

Diallyl trisulfide (DATS) is a secondary metabolite of allicin, a volatile organosulfur flavoring compound generated by the crushing of garlic. These compounds have various medicinal effects such as antiplatelet activity. In this study, we demonstrated for the first time the cellular mechanism involved in the inhibition of platelet aggregation by DATS and dipropyl trisulfide (DPTS), which is a saturated analogue of DATS. Washed murine platelets were incubated with these sulfides, and platelet aggregation was evaluated by light transmission aggregometry. The amount of reaction products produced by DATS, DPTS, and glutathione (GSH) was measured using liquid chromatography-mass spectrometry. Compared with DPTS, DATS potently inhibited platelet aggregation induced by thrombin, U46619, and collagen. N-Ethylmaleimide (NEM), which is commonly used to modify sulfhydryl groups, also suppressed platelet aggregation. The reactivity of DATS with GSH was higher than that of DPTS. These data suggested that DATS inhibited platelet aggregation through the reaction of sulfhydryl groups.

Tyrosinase from Penicillium chrysogenum: Characterization and application in phenol removal from aqueous solution.

The tyrosinase of Penicillium chrysogenum strain AUMC 14100 Accession No. MN219732 was purified to homogeneity and chemically modified by N-ethylmaleimide (NEM) and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride, DC). The inactivation of the purified enzyme obeyed pseudo-first-order reaction kinetics in the presence of NEM and DC (1-5 mM). The rate constants of the enzyme inactivation by NEM and DC were calculated to be 0.083 mol/min and 0.0013 mol/min, respectively. The recovery of enzyme activity by the protective effect of substrate indicates a non-specific modification of the active center. The order of tyrosinase inactivation kinetics and the substrate protection revealed the essentiality of sulfhydryl and lysyl residues in the enzyme active site and its role in the enzyme catalysis. The immobilized tyrosinase on alginate showed a gradual increase in residual activity over the immobilization time until the fourth hour. The desorptivity of tyrosinase was gradually raised with higher sodium dodecyl sulfate (SDS) concentrations. The immobilized enzyme retained about 70% of its original activity after 8 repeated cycles. Thus, immobilized tyrosinase of Penicillium chrysogenum removed 75% of phenol after 8 cycles and thus seems likely to be a good candidate for phenol removal in aqueous solution.

Progress in Membrane Fusion and Its Application in Drug Delivery

Abstract Membrane fusion plays an important role in regulating life activities of organisms. Intracellular membrane fusion plays a role in promoting the function of intracellular substances throughout the life cycle of an organism. The fusion of viruses and cell membranes may mark the end of life of an organism. However, due to the complexity of living cells, the interpretation of the results of organism research is often ambiguous. At present, only a few protein structures on the cell membrane, such as the soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNARE) protein family and its role in promoting membrane fusion, are gradually being explored. In order to better study the mechanism of membrane fusion, a related artificial model system has been established to simulate biofilm fusion, such as a membrane fusion research biological model established by using DNA strand modified liposomes and coiled-coil modified liposomes. This paper reviews the mechanism of SNARE-promoting membrane fusion and the established DNA and coiled-coil polypeptide-mediated membrane fusion studies, as well as the development of membrane fusion in drug delivery.

A ratiometric fluorescent nanoprobe consisting of ssDNA-templated silver nanoclustersfor detection of histidine/cysteine, and theconstruction of combinatorial logic circuits.

A ratiometric fluorescent nanoprobe consisting of ssDNA-templated silver nanoclusters (DNA-AgNCs) as dual fluorophore has been constructed for highly sensitive and selective detection of cystein (Cys) and histidine (His). The DNA-AgNCs displays dual emission in that photoexcitation at 470nm results in weak-green fluorescence peaking at 560nm, while excitation at 550nm gives strongred fluorescence with a peak at 595nm. It is found that copper ions (Cu2+) enhance the green fluorescence but quenche the red fluorescence. A ratiometric nanoprobe for Cys (or His) is designed that is based on the competitive interaction of Cys (or His), Cu2+ and DNA-AgNCs. Cys can be distinguished from His by adding Ni2+ as the masking agent, andr His can be distinguished from Cys by adding N-ethylmaleimide (NEM) as the masking agent, respectively. The limits of detection are 5.1 and 4.5nM for Cys and His, respectively. Furthermore, the ratiometric fluorescent nanoprobe is used for constructing combinatorial logic circuits in parallel, including OR//NOR and INHIBIT//IMPLICATION (INH//IMP). Graphical abstract Schematic representation of a ratiometric assay based on DNA-AgNCs with dual emission for detection of histidine/cysteine in serum sample. The sensing system are further used to construct combinatorial logic circuits.

Bacterial dynamin-like protein DynA mediates lipid and content mixing.

Many bacterial species contain dynamin-like proteins (DLPs). However, so far the functional mechanisms of bacterial DLPs are poorly understood. DynA in Bacillus subtilis is a 2-headed DLP, mediating nucleotide-independent membrane tethering in vitro and contributing to the innate immunity of bacteria against membrane stress and phage infection. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if DynA induces membrane full fusion, characterize its subunits in membrane fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer indicated that DynA could induce aqueous content mixing even in the absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow process, and it has phospholipid and membrane curvature preferences. The D1 part of DynA is crucial for membrane binding and fusion, whereas D2 subunit plays a role in facilitating membrane fusion. Surprisingly, digestion of DynA mediated an instant rise of content exchange, supporting the assumption that disassembly of DynA is a driving force for fusion-through-hemifusion. DynA is a rare example of a membrane fusion catalyst that lacks a transmembrane domain and hence sets this system apart from well-characterized fusion systems such as the soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes.-Guo, L., Bramkamp, M. Bacterial dynamin-like protein DynA mediates lipid and content mixing.

Purification and biochemical characterization of a new organic solvent-tolerant chitinase from Paenibacillus timonensis strain LK-DZ15 isolated from the Djurdjura Mountains in Kabylia, Algeria

A new extracellular chitinase (called ChiA-Pt70) was produced and purified from a newly isolated Paenibacillus timonensis strain LK-DZ15. The maximum chitinase activity recorded after 44-h of incubation at 30 °C was 11,500 U/mL. Pure enzyme was obtained after ammonium sulphate precipitation (40–70%) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 70,166.11 kDa. The sequence of the 25 NH2-terminal residues of the mature ChiA-70 showed high homology with Paenibacillus GH-18 chitinases family. Optimal activity was achieved at pH 4.5 and 80 °C. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB), 5,5′-dithio-bis-2-nitro benzoic acid (DTNB), and N-ethylmaleimide (NEM). Chitinase activity was high on colloidal chitin, chitin azure, glycol chitin, glycol chitosane, chitotriose, and chito-oligosaccharide while it did not hydrolyse chitibiose and amylose. Furthermore, thin-layer chromatography (TLC) analysis from enzymatic catalyzed hydrolysis of colloidal chitin showed that ChiA-Pt70 acted as an endo-splitting enzyme. Its Km and kcat values were 0.611 mg colloidal chitin/mL and 87,800 s−1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Mt45 from Melghiribacillus thermohalophilus strain Nari2AT, ChiA-Hh59 from Hydrogenophilus hirchii strain KB-DZ44, Chitodextrinase® from Streptomyces griseus, and N-acetyl-β-glucosaminidase® from Trichoderma viride. Therefore, ChiA-Pt70 exhibited remarkable biochemical properties suggesting that it is suitable for the enzymatic degradation of chitin.

Pharmacological analysis of transmission activation of two aphid-vectored plant viruses, turnip mosaic virus and cauliflower mosaic virus

Turnip mosaic virus (TuMV, family Potyviridae) and cauliflower mosaic virus (CaMV, family Caulimoviridae) are transmitted by aphid vectors. They are the only viruses shown so far to undergo transmission activation (TA) immediately preceding plant-to-plant propagation. TA is a recently described phenomenon where viruses respond to the presence of vectors on the host by rapidly and transiently forming transmissible complexes that are efficiently acquired and transmitted. Very little is known about the mechanisms of TA and on whether such mechanisms are alike or distinct in different viral species. We use here a pharmacological approach to initiate the comparison of TA of TuMV and CaMV. Our results show that both viruses rely on calcium signaling and reactive oxygen species (ROS) for TA. However, whereas application of the thiol-reactive compound N-ethylmaleimide (NEM) inhibited, as previously shown, TuMV transmission it did not alter CaMV transmission. On the other hand, sodium azide, which boosts CaMV transmission, strongly inhibited TuMV transmission. Finally, wounding stress inhibited CaMV transmission and increased TuMV transmission. Taken together, the results suggest that transmission activation of TuMV and CaMV depends on initial calcium and ROS signaling that are generated during the plant’s immediate responses to aphid manifestation. Interestingly, downstream events in TA of each virus appear to diverge, as shown by the differential effects of NEM, azide and wounding on TuMV and CaMV transmission, suggesting that these two viruses have evolved analogous TA mechanisms.

Enhancing the peroxidase-like activity of ficin by rational blocking thiol groups for colorimetric detection of biothiols

The peroxidase-like activity of ficin is relatively low, which limits its application. It was found that thiol groups of ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N-ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of ficin, ficin-NEM (ficin-N) and ficin-TCEP-NEM (ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of ficin-N and ficin-TN toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation by H2O2 was about 2.5-fold and 5-fold increase compared with ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of ficin-N (Km = 0.31) and ficin-TN (Km = 0.39) to H2O2 compared with ficin (Km = 0.58), and ficin-TN had the highest Kcat which increased by 6.5 times and 4.5 times for TMB and H2O2, respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of ficin. Comparing with ficin and ficin-N, ficin-TN had the widest detection range (0.01–16 μM) and the lowest detection limit (3 nM). The practical applications of ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and ficin-TN are promising to apply to the bioanalysis.

Characterization of the endonuclease activity of the replication-associated protein of beak and feather disease virus.

Beak and feather disease virus (BFDV) belongs to the family Circoviridae. A rolling-circle replication strategy based on a replication-associated protein (Rep) has been proposed for BFDV. The Rep gene of BFDV was expressed and purified, and it was shown to cleave short oligonucleotides containing the conserved nonanucleotide sequence found in the replication origin of circoviruses. This endonuclease activity was most efficient in the presence of the divalent metal ions Mg2+ and Mn2+. Rep proteins containing mutation in the ATPase/GTPase motifs and the 14FTLNN18, 61KKRLS65, 89YCSK92, and 170GKS172 motifs lacked endonuclease activity. The endonuclease activity was not affected by ATPase inhibitors, with the exception of N-ethylmaleimide (NEM), or by GTPase inhibitors, but it was decreased by treatment with the endonuclease inhibitor L-742001. Both the ATPase and GTPase activities were decreased by site-directed mutagenesis and deletion of the ATPase/GTPase and endonuclease motifs. The Rep protein was able to bind a double-stranded DNA fragment of P36 (dsP36) containing the stem-loop structure of the replication origin of BFDV. All of the Rep mutant proteins showed reduced ability to bind this fragment, suggesting that all the ATPase/GTPase and endonuclease motifs are involved in the binding. Other than NEM, all ATPase, GTPase, and endonuclease inhibitors inhibited the binding of the Rep protein to the dsP36 fragment. This is the first report describing the endonuclease activity of the Rep protein of BFDV.

Retrograde trafficking and quality control of yeast synaptobrevin, Snc1, are conferred by its transmembrane domain.

Synaptobrevin/vesicle-associated membrane protein 2 (VAMP2) is an essential soluble N-ethyl maleimide–sensitive factor attachment protein receptor (SNARE) protein that has been extensively studied in its role in synaptic vesicle fusion. However, sorting and trafficking of VAMP2 within the endosomal system is not well understood. Here, we use the yeast VAMP2 homologue Snc1 to investigate the pathways and signals required for endocytic trafficking. We identify two genetically distinct retrieval pathways from the endosomal system: a plasma membrane recycling pathway that requires the Rcy1 F-box protein and a retrograde pathway originating from the multivesicular/prevacuole endosome dependent on the Snx4-Atg20 sorting nexin complex. Lysine residues within the transmembrane domain of Snc1 are necessary for presentation of a Snx4-Atg20–dependent sorting signal located within its juxtamembrane region. Mutations of the transmembrane lysine residues ablate retrograde sorting and subject Snc1 to quality control via sorting into the degradative multivesicular endosome pathway. Degradative sorting requires lysine residues in the juxtamembrane region of Snc1 and is mediated by the Rsp5 ubiquitin ligase and its transmembrane adapters, Ear1 and Ssh4, which localize to endosome and vacuole membranes. This study shows that Snc1 is trafficked between the endosomal system and the Golgi apparatus via multiple pathways and provides evidence for protein quality control surveillance of a SNARE protein in the endo-vacuolar system.

AMADH inhibitor optimization and its effects on GABA accumulation in soybean sprouts under NaCl-CaCl2 treatment.

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was the suitable inhibitor for aminoaldehyde dehydrogenase (AMADH) compared with N-ethylmaleimide (NEE) and iodoacetamide (IAM). EDC exhibited the most obvious inhibition effect on AMADH activity, while its inhibition on glutamate decarboxylase (GAD) was insignificant. Compared with the control, AMADH activity reduced by 70.4% with 0.5mM EDC, and γ-aminobutyric acid (GABA) content declined by 44.3% in soybean sprouts at 4days of germination. AMADH activity reduced by 80.62, 67.61 and 72.02% in the 4-day sprouts with 1mM EDC under NaCl, CaCl2 and NaCl + CaCl2 treatment, respectively, and GABA content decreased by 43.56, 38.84 and 35.53%, respectively. EDC is a proper inhibitor for AMADH and it could be used to quantify the contribution of polyamine degradation pathway on GABA formation. In soybean sprouts, the presence of CaCl2 under NaCl stress decreased the contribution of polyamine degradation pathway on GABA accumulation.

Complete NEM Modification of Highly Reactive Cysteines Induces Full TRPA1 Activation

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, non‐selective, cationic channel that is expressed in the dorsal root ganglia, trigeminal ganglia, and vagal ganglia and is activated by harmful chemicals including pollutants and endogenous irritants produced during inflammation and oxidative stress. Many irritants activate TRPA1 via electrophilic modification of highly reactive cysteine residues located on the cytosolic side of the channel, however the underlying processes that precede activation are poorly understood. Our lab has identified four highly reactive cysteines on TRPA1 (thus 16 highly reactive cysteines per channel) that rapidly bind electrophiles: C273, C621, C665 and C1085. C621 was the most reactive and point mutation studies show that both C621 and C665 are required for activation. In this study, we use cell‐attached single‐channel patch clamp analyses of transiently expressed human TRPA1 (hTRPA1) in HEK293 cells to determine the relationship between irreversible electrophilic binding and channel activity. We recorded stochastic gating events of single channels in response to N‐ethylmaleimide (NEM) treatment. Our open probability (Po) analysis indicates that NEM evoke two distinct hTRPA1 activation profiles: (1) weak activation (low Po) throughout NEM treatment which does not increase with time; (2) weak activation which then suddenly and spontaneously progresses to full activation (near 100% Po). Open time histogram analyses suggest that the weak activation state evoked by NEM has similar open times to spontaneous hTRPA1 activation in control conditions; whereas the full activation state has significantly longer open times. We conclude that the weak and full hTRPA1 activation states by NEM are due to differences in progressive cysteine modification. Furthermore, we suggest that full activation is not the summation of multiple weak states, but is instead a separate activation state that can only be evoked by a particular complement of cysteine modifications.Support or Funding InformationThis research is funded by the National Heart, Lung and Blood Institute (5R01HL119802‐S1).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Matrix-induced transformation of arsenic species in seafoods

The paper presents a study on the matrix-induced transformation of arsenic (As) species spiked into seafoods. Sixteen arsenicals were individually spiked into samples of finfish, crustaceans and molluscs. The spiked samples were subjected to hot water extraction at 90 °C, and extracts were analyzed by high pressure liquid chromatography (HPLC) interfaced with inductively coupled plasma mass spectrometry (ICP-MS). Arsenate (As5+), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinate (DMA), monomethylarsonate (MMA), tetramethylarsonium ion (TMA) and trimethylarsoniopropionate (TMAP) remained intact in all the matrices. Whereas arsenite (As3+), dimethylarsinoyl acetate (DMAA), dimethylarsinoyl ethanol (DMAE), dimethylarsinoyl propionate (DMAP), trimethylarsine oxide (TMAO) and glycerol-, sulfonate-, sulfate- and phosphate-arsinoylribosides (arsenosugars 328, 392, 408 and 482, respectively) were transformed to other forms in most finfish and crustaceans. The transformation of the arsenicals was discovered to be induced by matrix thiols. While As3+ was bound to sulfhydryl groups, DMAA, DMAE, DMAP, TMAO and arsenosugars 392, 408 and 482 were thiolated through conversion of their arsinoyl (As=O) functionalities to arsinothioyl (As=S). The newly formed arsinothioyl compounds were characterized by HPLC-ICP-MS and electrospray ionization high-resolution tandem mass spectrometry (ESI-HR-MS/MS) paired with HPLC. The observed matrix-induced transformation of the arsenic species could be prevented by treating the samples (prior to spiking) with a thiol-selective blocking agent, N-ethylmaleimide (NEM).