The octadecaneuropeptide ODN prevents 6‐hydroxydopamine‐induced apoptosis of cerebellar granule neurons through a PKC‐MAPK‐dependent pathway

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6‐hydroxydopamine (6‐OHDA)‐induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central‐type benzodiazepine receptors (CBR) and a metabotropic receptor. Incubation of neurons with subnanomolar concentrations of ODN (10^?18 to 10^?12 M) inhibited 6‐OHDA‐evoked cell death in a concentration‐dependent manner. The effect of ODN on neuronal survival was abrogated by the metabotropic receptor antagonist, cyclo_1–8[DLeu⁵]OP, but not by a CBR antagonist. ODN stimulated polyphosphoinositide turnover and ERK phosphorylation in CGN. The protective effect of ODN against 6‐OHDA toxicity involved the phospholipase C/ERK MAPK transduction cascade. 6‐OHDA treatment induced an accumulation of reactive oxygen species, an increase of the expression of the pro‐apoptotic gene Bax, a drop of the mitochondrial membrane potential and a stimulation of caspase‐3 activity. Exposure of 6‐OHDA‐treated cells to ODN blocked all the deleterious effects of the toxin. Taken together, these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6‐OHDA‐induced oxidative stress and apoptotic cell death.     

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis is SOD1‐immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S‐nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up‐regulated, which generated high levels of nitric oxide (NO) and subsequently induced S‐nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH‐SY5Y cells with NO donor S‐nitrosocysteine triggered a dose‐dependent formation of S‐nitrosylated PDI. When mutant SOD1 was over‐expressed in SH‐SY5Y cells, the iNOS expression was up‐regulated, and NO generation was consequently increased. Furthermore, both S‐nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1^G93A. Blocking NO generation with the NOS inhibitor N‐nitro‐l ‐arginine attenuated the S‐nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO‐mediated S‐nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions

2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5′-dithio-bis-(2-nitrobenzoic acid) and ZnCl₂, which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H₂O₂. SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.     

FrnE,a Cadmium-Inducible Protein in Deinococcus radiodurans,Is Characterized as a Disulfide Isomerase Chaperone In Vitro and for Its Role in Oxidative Stress Tolerance In Vivo

Deinococcus radiodurans R1 exposed to a lethal dose of cadmium shows differential expression of a large number of genes, including frnE (drfrnE) and some of those involved in DNA repair and oxidative stress tolerance. The drfrnE::nptII mutant of D. radiodurans showed growth similar to that of the wild type, but its tolerance to 10 mM cadmium and 10 mM diamide decreased by ∼15- and ∼3-fold, respectively. These cells also showed nearly 6 times less resistance to gamma radiation at 12 kGy and ∼2-fold-higher sensitivity to 40 mM hydrogen peroxide than the wild type. In trans expression of drFrnE increased cytotoxicity of dithiothreitol (DTT) in the dsbA mutant of Escherichia coli. Recombinant drFrnE showed disulfide isomerase activity and could maintain insulin in its reduced form in the presence of DTT. While an equimolar ratio of wild-type protein could protect malate dehydrogenase completely from thermal denaturation at 42°C, the C22S mutant of drFrnE provided reduced protection to malate dehydrogenase from thermal inactivation. These results suggested that drFrnE is a protein disulfide isomerase in vitro and has a role in oxidative stress tolerance of D. radiodurans possibly by protecting the damaged cellular proteins from inactivation.     

Sialylneolacto-N-tetraose c (LSTc)-bearing Liposomal Decoys Capture Influenza A Virus

Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.     

RanBPM Protein Acts as a Negative Regulator of BLT2 Receptor to Attenuate BLT2-mediated Cell Motility

BLT2, a low affinity receptor for leukotriene B₄ (LTB₄), is a member of the G protein-coupled receptor family and is involved in many signal transduction pathways associated with various cellular phenotypes, including chemotactic motility. However, the regulatory mechanism for BLT2 has not yet been demonstrated. To understand the regulatory mechanism of BLT2, we screened and identified the proteins that bind to BLT2. Using a yeast two-hybrid assay with the BLT2 C-terminal domain as bait, we found that RanBPM, a previously proposed scaffold protein, interacts with BLT2. We demonstrated the specific interaction between BLT2 and RanBPM by GST pulldown assay and co-immunoprecipitation assay. To elucidate the biological function of the RanBPM-BLT2 interaction, we evaluated the effects of RanBPM overexpression or knockdown. We found that BLT2-mediated motility was severely attenuated by RanBPM overexpression and that knockdown of endogenous RanBPM by shRNA strongly promoted BLT2-mediated motility, suggesting a negative regulatory function of RanBPM toward BLT2. Furthermore, we observed that the addition of BLT2 ligands caused the dissociation of BLT2 and RanBPM, thus releasing the negative regulatory effect of RanBPM. Finally, we propose that Akt-induced BLT2 phosphorylation at residue Thr³⁵⁵, which occurs after the addition of BLT2 ligands, is a potential mechanism by which BLT2 dissociates from RanBPM, resulting in stimulation of BLT2 signaling. Taken together, our results suggest that RanBPM acts as a negative regulator of BLT2 signaling to attenuate BLT2-mediated cell motility.     

Testing the Sulfotransferase Molecular Pore Hypothesis

Human cytosolic sulfotransferases (SULTs) regulate the activities of hundreds of signaling metabolites via transfer of the sulfuryl moiety (-SO₃) from activated sulfate (3′-phosphoadenosine 5′-phosphosulfate) to the hydroxyls and primary amines of xeno- and endobiotics. How SULTs select substrates from the scores of competing ligands present in a cytosolic milieu is an important issue in the field. Selectivity appears to be sterically controlled by a molecular pore that opens and closes in response to nucleotide binding. This point of view is fostered by structures showing nucleotide-dependent pore closure and the fact that nucleotide binding induces an isomerization that restricts access to the acceptor-binding pocket. Molecular dynamics models underscore the importance of pore isomerization in selectivity and predict that specific molecular linkages stabilize the closed pore in response to nucleotide binding. To test the pore model, these linkages were disrupted in SULT2A1 via mutagenesis, and the effects on selectivity were determined. The mutations uncoupled nucleotide binding from selectivity and produced enzymes that no longer discriminated between large and small substrates. The mutations did not affect the affinity or turnover of small substrates but resulted in a 183-fold gain in catalytic efficiently toward large substrates. Models predict that an 11-residue “flap” covering the acceptor-binding pocket can open and admit large substrates when nucleotide is bound; a mutant structure demonstrated that this is so. In summary, the model was shown to be a robust, accurate predictor of SULT structure and selectivity whose general features will likely apply to other members of the SULT family.     

Wiskott-Aldrich Syndrome Protein (WASp) Controls the Delivery of Platelet Transforming Growth Factor-β1

Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp^−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp^−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp^−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.