Characterization of monogalactosyldiacylglycerol synthases in Gracilariopsis lemaneiformis and their potential roles in the fading of the thallus.

Membrane lipids play essential roles in regulating physiological properties in higher plants and algae. Monogalactosyldiacylglycerol (MGDG) is a major thylakoid membrane lipid, and it is an important source of polyunsaturated fatty acids for cells, plays a key role in the biogenesis of plastids, and maintains the function of the photosynthetic machinery. Several studies have indicated that the knockdown of MGDG synthase results in membrane lipid remodeling, albino seedlings, and changes in photosynthetic performance. However, the effects of MGDG synthase (MGD) inhibitors on lipids in macroalgae have not yet been clarified. Here, we characterized the effects of MGD inhibitors (ortho-phenanthroline and N-ethylmaleimide) on the composition of the fatty acids observed in MGDG and digalactosyldiacylglycerol (DGDG) in Gracilariopsis lemaneiformis using electrospray ionization-mass spectrometry. The most abundant MGDG species contained 16:0/18:1 (sn-1/sn-2) fatty acids, and the most dominant DGDG species contained 20:5/16:0 (sn-1/sn-2) fatty acids. Measurements of photosynthetic pigments and photosynthetic parameters revealed that photosynthesis of G. lemaneiformis was impaired. Principal component analysis and Spearman's correlation analysis revealed interactions between specific MGDG structural composition patterns and key metabolites involved in photosynthesis, indicating that 20:4/16:0 (sn-1/sn-2) MGDG and 16:0/18:1 (sn-1/sn-2) MGDG affect the structure and function of phycobilisomes and thus the color of G. lemaneiformis. Three genes (GlMGD1, GlMGD2, and GlMGD3) were cloned and identified. The addition of N-ethylmaleimide to G. lemaneiformis did not affect the abundance of GlMGD mRNA, and the abundance of transcripts was significantly decreased by ortho-phenanthroline.

Apicoplast biogenesis mediated by ATG8 requires the ATG12-ATG5-ATG16L and SNAP29 complexes in Toxoplasma gondii.

In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in Toxoplasma and Plasmodium, ATG12 interacts with ATG5 in a non-covalent manner, in contrast to the covalent interaction in most organisms. However, it remained unknown whether apicomplexan parasites have functional orthologs of ATG16L1, a protein that is essential for the function of the covalent ATG12-ATG5 complex in vivo in other organisms. Furthermore, the mechanism used by the autophagy machinery to maintain the apicoplast is unclear. We report that the ATG12-ATG5-ATG16L complex exists in Toxoplasma gondii (Tg). This complex is localized on isolated structures at the periphery of the apicoplast dependent on TgATG16L. Inducible depletion of TgATG12, TgATG5, or TgATG16L caused loss of the apicoplast and affected parasite growth. We found that a putative soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein, synaptosomal-associated protein 29 (TgSNAP29, Qbc SNARE), is required to maintain the apicoplast in T. gondii. TgSNAP29 depletion disrupted TgATG8 localization at the apicoplast. Additionally, we identified a putative ubiquitin-interacting motif-docking site (UDS) of TgATG8. Mutation of the UDS site abolished TgATG8 localization on the apicoplast but not lipidation. These findings suggest that the TgATG12-TgATG5-TgATG16L complex is required for biogenesis of the apicoplast, in which TgATG8 is translocated to the apicoplast via vesicles in a SNARE -dependent manner in T. gondii.Abbreviations: AID: auxin-inducible degron; CCD: coiled-coil domain; HFF: human foreskin fibroblast; IAA: indole-3-acetic acid; LAP: LC3-associated phagocytosis; NAA: 1-naphthaleneacetic acid; PtdIns3P: phosphatidylinositol-3-phosphate; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; UDS: ubiquitin-interacting motif-docking site; UIM: ubiquitin-interacting motif.

Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity.

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O2). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila.

The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes.Abbreviations: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; Atg: autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region.

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.

Role of SNAREs in Atopic Dermatitis-Related Cytokine Secretion and Skin-Nerve Communication.

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptors in atopic dermatitis (AD) is unknown. This study identifies the function of soluble N-ethylmaleimide sensitive factor attachment protein receptor in AD-related cytokine secretion and epidermis-nerve communication. Herein, we report that various cytokines were simultaneously upregulated and coreleased in innate immunity-activated primary human keratinocytes. AD-related cytokines thymic stromal lymphopoietin, endothelin-1, and inflammatory tumor necrosis factor-α activated distinct but overlapping sensory neurons. Tumor necrosis factor-α potentiated thymic stromal lymphopoietin-induced Ca2+-influx, whereas endothelin-1 caused itch-selective B-type natriuretic peptide release. In primary human keratinocytes, B-type natriuretic peptide upregulated genes promoting dermatological and neuroinflammatory diseases and conditions. VAMP3, SNAP-29, and syntaxin 4 proved important in driving cytokine release from primary human keratinocytes. Depletion of VAMP3 inhibited nearly all the cytokine release including thymic stromal lymphopoietin and endothelin-1. Accordingly, VAMP3 co-occurred with endothelin-1 in the skins of patients with AD. Our study pinpoints the pivotal role of soluble N-ethylmaleimide sensitive factor attachment protein receptors in mediating cytokine secretion related to AD. VAMP3 is identified as a suitable target for developing broad-spectrum anticytokine therapeutics for controlling itch and atopic skin inflammation.

Phosphoregulation of the intracellular termini of K+-Cl- cotransporter 2 (KCC2) enables flexible control of its activity.

The pivotal role of K+-Cl- cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five bona fide phosphosites Ser31, Thr34, Ser932, Thr999, and Thr1008 was investigated by the use of alanine and aspartate mutants. Tl+-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent. Increased activity was due to changes in intrinsic activity, as it was not caused by increased cell-surface abundance. Substitutions of Ser31, Thr34, or Thr999 had no effect. Additionally, we show that the indirect actions of the known KCC2 activators staurosporine and N-ethylmaleimide (NEM) involved multiple phosphosites. S31D, T34A, S932A/D, T999A, or T1008A/D abrogated staurosporine mediated stimulation, and S31A, T34D, or S932D abolished NEM-mediated stimulation. This demonstrates for the first time differential effects of staurosporine and NEM on KCC2. In addition, the staurosporine-mediated effects involved both KCC2 phosphorylation and dephosphorylation with Ser932 and Thr1008 being bona fide target sites. In summary, our data reveal a complex phosphoregulation of KCC2 that provides the transporter with a toolbox for graded activity and integration of different signaling pathways.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1).

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation.

K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.

Calcium Channel Opening Rather than the Release of ATP Causes the Apoptosis of Osteoblasts Induced by Overloaded Mechanical Stimulation.

BACKGROUND: Stress fracture is one of the most common overuse injuries in athletes. Overloaded mechanical stimulation is an important factor affecting stress fractures, but the mechanism is unclear. METHODS: MC3T3-E1 cells and a polycaprolactone (PCL) scaffold were co-cultured, and finite element analysis (FEA) was used to analyze the load-carrying capability. Cell proliferation was investigated with CCK-8 assays. An alkaline phosphatase (AKP) activity assay was used to evaluate cell differentiation. Cell apoptosis was analyzed using Hoechst/ PI double-labeling, Caspase-3 activity and lactate dehydrogenase (LDH) activity assays. Realtime PCR and Western blotting were used to examine the gene and protein expression, respectively, of Caspase-3 and Caspase-9. Assays of the intracellular calcium with fluorescent probe technique and extracellular ATP with fluorometric assay kit were used to analyze the changes in the intracellular calcium concentration induced by calcium channel opening and the release of ATP, respectively, at different operation times. RESULTS: When the apparent strain reached 10000 µÎµ, the strain scope of fber at levels greater than 4000 µÎµ was 60%. Overloading for 4 days and operation times of 0.5 h and 2 h increased the cell number and AKP secretion. However, apoptosis genes were activated at the same time, and the operation time of 2 h had a significantly greater effect than 0.5 h. At 8 days, the cell numbers were greater for the operation time of 0.5 h than for 2 h, and the 2-h groups had the fastest apoptosis rate. Overloading for 1 day increased intracellular calcium levels and ATP release. The increase in intracellular calcium could be blocked by the addition of N-ethylmaleimide (NEM) or Hank's medium. Overloading for 8 days increased intracellular calcium levels but decreased extracellular ATP, and verapamil blocked the increase in intracellular calcium. CONCLUSION: We found that a simultaneous 'double effect' on osteoblasts was induced by overloading, which promoted cell proliferation, differentiation and apoptosis. Short-term overloading could open the cell membrane calcium channels and release calcium stores to elevate intracellular calcium levels, thereby promoting the proliferation and differentiation of cells to a greater extent than the effect of apoptosis. For long-term overloading, calcium channel opening in the membrane could lead to overloading of intracellular calcium levels, inducing an apoptosis effect that is greater than the effect on proliferation and differentiation.

N-Ethylmaleimide Dissociates α7 ACh Receptor from a Complex with NSF and Promotes Its Delivery to the Presynaptic Membrane.

N-Ethylmaleimide (NEM)-sensitive factor (NSF) associates with soluble NSF attachment protein (SNAP), that binds to SNAP receptors (SNAREs) including syntaxin, SNAP25, and synaptobrevin. The complex of NSF/SNAP/SNAREs plays a critical role in the regulation of vesicular traffic. The present study investigated NEM-regulated α7 ACh receptor translocation. NSF associated with ß-SNAP and the SNAREs syntaxin 1 and synaptobrevin 2 in the rat hippocampus. NSF also associated with the α7 ACh receptor subunit, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and the γ-aminobutyric acid A (GABAA) receptor γ2 subunit. NEM, an inhibitor of NSF, significantly dissociated the α7 ACh receptor subunit from a complex with NSF and increased cell surface localization of the receptor subunit, but such effect was not obtained with the GluA1, GluA2 or γ2 subunits. NEM, alternatively, dissociated synaptobrevin 2 from an assembly of NSF/ß-SNAP/syntaxin 1/synaptobrevin 2. NEM significantly increased the rate of nicotine-triggered AMPA receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, in rat hippocampal slices. The results of the present study indicate that NEM releases the α7 ACh receptor subunit and synaptobrevin 2 from an assembly of α7 ACh receptor subunit/NSF/ß-SNAP/syntaxin 1/synaptobrevin 2, thereby promoting delivery of the α7 ACh receptor subunit to presynaptic membrane.

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

BACKGROUND: Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. RESULTS: Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. CONCLUSIONS: Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Reaction of Human Cd7metallothionein and N-Ethylmaleimide: Kinetic and Structural Insights from Electrospray Ionization Mass Spectrometry.

The reaction of cadmium-binding human metallothionein-2A (Cd7MT) and N-ethylmaleimide (NEM) is investigated by electrospray ionization-ion mobility-mass spectrometry (ESI IM-MS). MS provides a direct measure of the distribution of the kinetic intermediates as the reaction proceeds and provides new insights into the relative kinetic stability of the individual metal-thiolate bonds in Cd7MT. The rate constants for the various metal-retaining intermediates (Cd(i), intermediate with i Cd²âº ions attached) differ by >3 orders of magnitude: Cd4< Cd3< Cd2< Cd1∼ Cd6 < Cd7 < Cd5. The reaction is viewed as a two-component cooperative process, rapid loss of three Cd²âº ions followed by slow loss of the remaining four Cd²âº ions, and Cd4NEM10MT was observed as the least reactive intermediate during the entire displacement process. "MS-CID-IM-MS", a top-down approach that provides two-dimensional dispersion (size to charge by IM; mass to charge by MS) of the CID fragment ions, was used for direct analysis of the kinetic intermediate [Cd4NEM10MT]5⁺ ion. The results provide direct evidence that the four Cd²âº ions located in the α-domain are retained, indicative of the greater kinetic stability for the α-domain. Further, the mapping of the alkylation sites in the [Cd4NEM10MT]5⁺ ion reveals that not only the nine cysteines in the ß-domain but Cys33 in the α-domain is selectively labeled. The kinetic lability of the Cd-Cys33 bond is unexpected. The structural and functional implications of these findings are discussed.

Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids.

Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2-carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.

Efficacy as an intrinsic property of the M(2) muscarinic receptor in its tetrameric state.

Muscarinic and other G protein-coupled receptors exhibit an agonist-specific heterogeneity that tracks efficacy and commonly is attributed to an effect of the G protein on an otherwise homogeneous population of sites. To examine this notion, M2 muscarinic receptors were purified from Sf9 cells as monomers devoid of G protein and reconstituted as tetramers in phospholipid vesicles. In assays with N-[(3)H]methylscopolamine, seven agonists revealed a dispersion of affinities indicative of two or more classes of sites. Unlabeled N-methylscopolamine and the antagonist quinuclidinylbenzilate recognized one class of sites; atropine recognized two classes with a preference that was the opposite of that of agonists, as indicated by the effects of N-ethylmaleimide. The data were inconsistent with an explicit model of constitutive asymmetry within a tetramer, and the fit improved markedly upon the introduction of cooperative interactions (P < 0.00001). Purified monomers appeared to be homogeneous or nearly so to all ligands except the partial agonists pilocarpine and McN-A-343, where heterogeneity emerged from intramolecular cooperativity between the orthosteric site and an allosteric site. The breadth of each dispersion was quantified empirically as the area between the fitted curve for two classes of sites and the theoretical curve for a single class of lower affinity, which approximates the expected effect of GTP if a G protein were present. The areas measured for 10 ligands at reconstituted tetramers correlated with similar measures of heterogeneity and with intrinsic activities reported previously for binding and response in natural membranes (P ≤ 0.00002). The data suggest that the GTP-sensitive heterogeneity typically revealed by agonists at M2 receptors is intrinsic to the receptor in its tetrameric state. It exists independently of the G protein, and it appears to arise at least in part from cooperativity between linked orthosteric sites.

Identification of the redox partners of ERdj5/JPDI, a PDI family member, from an animal tissue.

ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue.

The role of cysteines and histidins of the norepinephrine transporter.

The thiol reagent N-ethylmaleimide (NEM) is known to inhibit irreversibly ligand binding by the norepinephrine transporter (NET), while the simultaneous presence of NET substrates or ligands protects from this inhibition. Therefore, cysteine residues located within the substrate binding pocket of the NET were assumed to play an important role in ligand binding. To examine which (if any) of the 10 cysteines (Cys) of the human (h) NET might be involved in transport and/or binding function, we mutated all hNET cysteines to alanine. Using transfected HEK293 cells we studied NEM effects on the hNET with respect to [(3)H]nisoxetine binding. Two cysteines (Cys176 and Cys185) within the extracellular loop of the NET have been proposed to form a disulfide bond. We could demonstrate that this is of crucial importance as corresponding hNET mutants, in which these cysteines have been replaced, showed a lack of plasma membrane expression. However, due to their oxidized state in the native NET protein, Cys176 and Cys185 may not be targets for NEM. All other Cys-to-Ala hNET mutants were fully active and showed no change in inhibition of [(3)H]nisoxetine binding by NEM. These observations clearly exclude cysteines as being involved in hNET ligand binding. Since NEM also interacts with histidin (His), we mutated all 13 histidins of the hNET to alanine and examined the NET mutants in functional and binding assays. His222 within the large extracellular loop of the transporter was identified as an interaction partner of NEM since in the corresponding hNET mutant NEM exhibited a significantly reduced inhibitory potency. Furthermore, we could show that histidins in position 296, 370 and 372 are important for nisoxetine binding, while His220, 441, 598 and 599 are crucial for plasma membrane expression of the hNET.

Reversible inactivation of dihydrolipoamide dehydrogenase by mitochondrial hydrogen peroxide.

Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.

A yeast metabolite extraction protocol optimised for time-series analyses.

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.

Pathways of cadmium fluxes in the root of the halophyte Suaeda salsa.

Halophyte plants offer a greater potential for phytoremediation research for reducing the levels of toxic metals from saline soils than salt sensitive plants. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd(2+) fluxes at different regions of the root apex of Suaeda salsa. The Cd(2+) influx in the rhizosphere was greatest near the root tip (within 150µm of the tip). The results indicated that Cd(2+) influx into roots was significantly suppressed by the pre-treatment or in the presence of two kinds of Ca(2+) channel blockers; LaCl(3) and verapamil. The Cd(2+) influx was also reduced by N-ethylmaleimide, a thiol blocker. Cd content determination and labeling of Cd using fluorescent dye support our conclusion. The results of this study provide a more stable theoretical basis for the phytoremediation of Cd contamination in saline soils of coastal zones.