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Trigeminal neuralgia, historically dubbed the “suicide disease,” is an exceedingly painful neurologic condition characterized by sudden episodes of intense facial pain. Unfortunately, the only U.S. Food and Drug Administration (FDA)–approved medication for trigeminal neuralgia carries substantial side effects, with many patients requiring surgery. Here, we identify the NRF2 transcriptional network as a potential therapeutic target. We report that cerebrospinal fluid from patients with trigeminal neuralgia accumulates reactive oxygen species, several of which directly activate the pain-transducing channel TRPA1. Similar to our patient cohort, a mouse model of trigeminal neuropathic pain also exhibits notable oxidative stress. We discover that stimulating the NRF2 antioxidant transcriptional network is as analgesic as inhibiting TRPA1, in part by reversing the underlying oxidative stress. Using a transcriptome-guided drug discovery strategy, we identify two NRF2 network modulators as potential treatments. One of these candidates, exemestane, is already FDA-approved and may thus be a promising alternative treatment for trigeminal neuropathic pain.

Significance Cocaine is a monoamine transport inhibitor. Current models attributing pharmacologic actions of cocaine to inhibiting the activity of the amine transporters alone failed to translate to the clinic. Cocaine inhibition of the dopamine, serotonin, and norepinephrine transporters is relatively weak, suggesting that blockade of the amine transporters alone cannot account for the actions of cocaine, especially at low doses. There is evidence for significantly more potent actions of cocaine, suggesting the existence of a high-affinity receptor(s) for the drug. Identifying and characterizing such receptors will deepen our understanding of cocaine pharmacologic actions and pave the way for therapeutic development. Here we identify a high-affinity cocaine binding site associated with BASP1 that is involved in mediating the drug’s psychotropic actions.

Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity, the neuronal process of adapting to diverse and variable inputs, depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR -null ( BVR −/− ) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. RNA sequencing, biochemistry, and pathway analyses suggested that these deficits were mediated through the loss of focal adhesion signaling at both the transcriptional and biochemical level in the hippocampus. Independently of its catalytic function, BVR acted as a bridge between the primary focal adhesion signaling kinases FAK and Pyk2 and the effector kinase Src. Without BVR, FAK and Pyk2 did not bind to and stimulate Src, which then did not phosphorylate the N -methyl- d -aspartate (NMDA) receptor, a critical posttranslational modification for synaptic plasticity. Src itself is a molecular hub on which many signaling pathways converge to stimulate NMDAR-mediated neurotransmission, thus positioning BVR at a prominent intersection of synaptic signaling.

Alzheimer’s disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFTs), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFTs, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H(2)S) is dysregulated during aging. H(2)S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine γ-lyase (CSE), the biosynthetic enzyme for H(2)S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H(2)S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3β (GSK3β). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H(2)S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD.

Synapses are complex bridges that connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. However, they must remain dynamic in order to adapt to changing inputs. Here, we report that the enzyme biliverdin reductase (BVR) physically links together key molecules in focal adhesion signaling at the synapse. In challenging mice with a battery of neurocognitive tasks, we first discover that BVR null (BVR-/-) mice exhibit profound deficits in learning and memory. We uncover that these deficits may be explained by a loss of focal adhesion signaling that is both transcriptionally and biochemically disrupted in BVR-/- hippocampi. We learn that BVR mediates focal adhesion signaling by physically bridging the initiatory kinases FAK/Pyk2 to the effector kinase Src. Activated Src normally promotes synaptic plasticity by phosphorylating the N-methyl-D-aspartate (NMDA) receptor, but FAK/Pyk2 are unable to bind and stimulate Src without BVR. Src itself is a molecular hub upon which many signaling pathways converge in order to stimulate NMDA neurotransmission, positioning BVR at a prominent intersection of synaptic signaling.

Cocaine is a behavioral stimulant with substantial abuse potential related to its positively rewarding actions1,2. Cocaine inhibits the reuptake inactivation of neurotransmitters such as dopamine, serotonin, and norepinephrine at high nanomolar to low micromolar concentrations2. There is evidence for substantially more potent influences of cocaine. For instance, Calligaro and Eldefrawi reported binding of [3H]cocaine to brain membranes with a dissociation constant of about 16 nM3. At 10 nM concentration, cocaine elicits environmental place conditioning in planarians4. Furthermore, 1nM cocaine enhances dopamine D2 receptor agonist-mediated signaling5. Inhibition of amine reuptake by cocaine is substantially less potent than some of these high affinity actions. Thus, evidence for a specific, high affinity receptor for cocaine that mediates its behavioral actions has been lacking. We now report high affinity binding of cocaine to the membrane-associated brain acid soluble protein-1 (BASP1) with a Kd of 7 nM. Knocking down BASP1 in the striatum inhibits [3H]cocaine binding to striatal synaptosomes. Depletion of BASP1 in the nucleus accumbens diminishes locomotor stimulation, acquisition, and expression of locomotor sensitization to cocaine. Our findings indicate that BASP1 is a pharmacologically relevant receptor for cocaine and a putative therapeutic target for psychostimulant addiction.

Immunometabolism as therapeutic target Dimethyl fumarate (DMF) is an immunomodulatory compound used to treat multiple sclerosis and psoriasis whose mechanisms of action remain only partially understood. Kornberg et al. found that DMF and its metabolite, monomethyl fumarate, succinate the glycolytic enzyme GAPDH (see the Perspective by Matsushita and Pearce). After DMF treatment, GAPDH was inactivated, and aerobic glycolysis was down-regulated in both myeloid and lymphoid cells. This resulted in down-modulated immune responses because inflammatory immune-cell subsets require aerobic glycolysis. Thus, metabolism can serve as a viable therapeutic target in autoimmune disease. Science , this issue p. 449 ; see also p. 377

ATM drives DNA repair by phosphorylating the histone variant H2AX. While ATM mutations elicit prominent neurobehavioral phenotypes, neural roles for H2AX have been elusive. We report impaired motor learning and balance in H2AX-deficient mice. Mitigation of reactive oxygen species (ROS) with N-acetylcysteine (NAC) reverses the behavioral deficits. Mouse embryonic fibroblasts deficient for H2AX exhibit increased ROS production and failure to activate the antioxidant response pathway controlled by the transcription factor NRF2. The NRF2 targets GCLC and NQO1 are depleted in the striatum of H2AX knockouts, one of the regions most vulnerable to ROS-mediated damage. These findings establish a role for ROS in the behavioral deficits of H2AX knockout mice and reveal a physiologic function of H2AX in mediating influences of oxidative stress on NRF2-transcriptional targets and behavior., H2AX is a histone variant with an essential function in DNA double-strand break repair and genome stability. Here, Weyemi and colleagues show that loss of neuronal H2AX leads to locomotor dysfunction and alteration in oxidative stress response.

Inositol hexakisphosphate kinase 1 (IP6K1), which generates 5-diphosphoinositol pentakisphosphate (5-IP7), physiologically mediates numerous functions. We report that IP6K1 deletion leads to brain malformation and abnormalities of neuronal migration. IP6K1 physiologically associates with α-actinin and localizes to focal adhesions. IP6K1 deletion disrupts α-actinin’s intracellular localization and function. The IP6K1 deleted cells display substantial decreases of stress fiber formation and impaired cell migration and spreading. Regulation of α-actinin by IP6K1 requires its kinase activity. Deletion of IP6K1 abolishes α-actinin tyrosine phosphorylation, which is known to be regulated by focal adhesion kinase (FAK). FAK phosphorylation is substantially decreased in IP6K1 deleted cells. 5-IP7, a product of IP6K1, promotes FAK autophosphorylation. Pharmacologic inhibition of IP6K by TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] recapitulates the phenotype of IP6K1 deletion. These findings establish that IP6K1 physiologically regulates neuronal migration by binding to α-actinin and influencing phosphorylation of both FAK and α-actinin through its product 5-IP7.

Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.

Rationale: Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. Objective: We have investigated the role of IPMK in regulating angiogenesis. Methods and Results: Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel–Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood–brain barrier and enhances brain blood vessel permeability. Conclusions: IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α.

Cystathionine γ-lyase, which is responsible for the production of cysteine, is decreased in the striatum and cortex of mouse models of Huntington’s disease and in patients with Huntington’s disease, and cysteine supplementation in diet and drinking water partly rescues the phenotype and the diminished longevity of the mouse model. Huntington's disease is associated with polyglutamine expansion in the gene encoding huntingtin. Mutant huntingtin is expressed throughout the brain and rest of the body, but the striatum is the most affected brain region. Here it is shown that the enzyme cystathionine γ-lyase (CSE), responsible for cysteine biosynthesis, is decreased in the striatum and cortex of both mouse models and Huntington's disease patients. Mutant huntingtin inhibits the transcriptional activator Sp1, resulting in decreased CSE transcription. Cysteine supplementation in diet and drinking water partially rescues the phenotype and the diminished longevity in the mouse model, suggesting that cysteine supplementation might be beneficial for Huntington's disease patients. Huntington’s disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes1. Huntington’s disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington’s disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity2. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington’s disease tissues, which may mediate Huntington’s disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington’s disease tissues and in intact mouse models of Huntington’s disease, suggesting therapeutic potential.

Summary Bilirubin is one of the most frequently measured metabolites in medicine, yet its physiologic roles remain unclear. Bilirubin can act as an antioxidant in vitro, but whether its redox activity is physiologically relevant is unclear because many other antioxidants are far more abundant in vivo. Here, we report that depleting endogenous bilirubin renders mice hypersensitive to oxidative stress. We find that mice lacking bilirubin are particularly vulnerable to superoxide (O2⋅−) over other tested reactive oxidants and electrophiles. Whereas major antioxidants such as glutathione and cysteine exhibit little to no reactivity toward O2⋅−, bilirubin readily scavenges O2⋅−. We find that bilirubin's redox activity is particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging O2⋅− during NMDA neurotransmission. Bilirubin's unique redox activity toward O2⋅− may underlie a prominent physiologic role despite being significantly less abundant than other endogenous and exogenous antioxidants.

SummarymTOR complex 1 (mTORC1; mammalian target of rapamycin [mTOR] in complex with raptor) is a key regulator of protein synthesis and cell growth in response to nutrient amino acids. Here we report that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid kinase activities, regulates amino acid signaling to mTORC1. This regulation is independent of IPMK's catalytic function, instead reflecting its binding with mTOR and raptor, which maintains the mTOR-raptor association. Thus, IPMK appears to be a physiologic mTOR cofactor, serving as a determinant of mTORC1 stability and amino acid-induced mTOR signaling. Substances that block IPMK-mTORC1 binding may afford therapeutic benefit in nutrient amino acid-regulated conditions such as obesity and diabetes.

Inositol pyrophosphates have been implicated in numerous biological processes. Inositol hexakisphosphate kinase-2 (IP6K2), which generates the inositol pyrophosphate, diphosphoinositol pentakisphosphate (IP7), influences apoptotic cell death. The tumor suppressor p53 responds to genotoxic stress by engaging a transcriptional program leading to cell-cycle arrest or apoptosis. We demonstrate that IP6K2 is required for p53-mediated apoptosis and modulates the outcome of the p53 response. Gene disruption of IP6K2 in colorectal cancer cells selectively impairs p53-mediated apoptosis, instead favoring cell-cycle arrest. IP6K2 acts by binding directly to p53 and decreasing expression of proarrest gene targets such as the cyclin-dependent kinase inhibitor p21.

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K39s role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function.

Inositol pyrophosphates physiologically regulate vesicular endocytosis, ribosomal disposition, and directly phosphorylate proteins. Here we demonstrate roles in cell death and regulation of telomere length. Lethal actions of wortmannin and caffeine are selectively abolished in yeast mutants that cannot synthesize inositol pyrophosphates. Wortmannin and caffeine appear to act through the phosphoinositide 3-kinase-related protein kinases Tel1 and Mec1, known regulators of telomere length. Inositol pyrophosphates physiologically antagonize the actions of these kinases, which is demonstrated by the fact that yeast mutants with reduced or elevated levels of inositol pyrophosphates, respectively, display longer and shorter telomeres.

The specificity of the reactions of nitric oxide (NO) with its neuronal targets is determined in part by the precise localizations of neuronal NO synthase (nNOS) within the cell. The targeting of nNOS is mediated by adapter proteins that interact with its PDZ domain. Here, we show that the nNOS adapter protein, CAPON, interacts with synapsins I, II, and III through an N-terminal phosphotyrosine-binding domain interaction, which leads to a ternary complex comprising nNOS, CAPON, and synapsin I. The significance of this ternary complex is demonstrated by changes in subcellular localization of nNOS in mice harboring genomic deletions of both synapsin I and synapsin II . These results suggest a mechanism for specific actions of NO at presynaptic sites.

Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations. The coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95, through a unique PDZ–PDZ domain interaction between PSD95 and nNOS. Here, we report the identification of a novel nNOS-associated protein, CAPON, which is highly enriched in brain and has numerous colocalizations with nNOS. CAPON interacts with the nNOS PDZ domain through its C terminus. CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells. CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes.

An error in immunoblotting data in Fig. 5C is corrected.

The tumor suppressor protein p53 is a critical stress response transcription factor that induces the expression of genes leading to cell cycle arrest, apoptosis, and tumor suppression. We found that mammalian inositol polyphosphate multikinase (IPMK) stimulated p53-mediated transcription by binding to p53 and enhancing its acetylation by the acetyltransferase p300 independently of its inositol phosphate and lipid kinase activities. Genetic or RNA interference (RNAi)-mediated knockdown of IPMK resulted in decreased activation of p53, decreased recruitment of p53 and p300 to target gene promoters, abrogated transcription of p53 target genes, and enhanced cell viability. Additionally, blocking the IPMK-p53 interaction decreased the extent of p53-mediated transcription. These results suggest that IPMK acts as a transcriptional coactivator for p53 and that it is an integral part of the p53 transcriptional complex facilitating cell death.

We have stably expressed cDNA for the rat brain Ca2+ sensing receptor in Chinese hamster ovary cells. Stimulation of phosphatidylinositol hydrolysis and arachidonic acid (AA) release displayed markedly cooperative responses to Ca2+ with Hill coefficients of 4-5. Both phosphatidylinositol and AA responses were not detected below a threshold of 1.5 mM Ca2+. Mg2+ behaved as a partial agonist with only half the maximal inositol phosphate and AA responses displayed by Ca2+ and with a more shallow concentration-response slope. The potency of Mg2+ in augmenting inositol phosphate and AA responses, in the presence of 1.5 mM Ca2+, implies that serum Mg2+ concentrations attained in clinical conditions will influence the Ca2+-sensing receptor.

We have molecularly cloned a calcium sensing receptor (CaSR) from a rat striatal cDNA library. Rat CaSR displays 92% overall homology to its bovine counterpart with seven putative transmembrane domains characteristic of the superfamily of guanine nucleotide-binding proteins and significant homology with the metabotropic glutamate receptors. Northern blot analysis reveals two transcripts in thyroid, kidney, lung, ileum, and pituitary. In brain highest regional expression of the RNA occurs in the hypothalamus and the corpus striatum. Immunohistochemistry reveals discrete punctate localizations throughout the brain that appear to be associated with nerve terminals. No staining is evident in cell bodies of neurons or glia. Cerebral arteries display an intense network of CaSR immunoreactive fibers associated with vessel innervation. CaSR on nerve terminal membranes may regulate neurotransmitter disposition in response to Ca2+ levels in the synaptic space.

Using antibodies that react selectively with peptide sequences unique to endothelial nitric oxide synthase (eNOS), we demonstrate localizations to neuronal populations in the brain. In some brain regions, such as the cerebellum and olfactory bulb, eNOS and neuronal NOS (nNOS) occur in the same cell populations, though in differing proportions. In the hippocampus, localizations of the two enzymes are strikingly different, with eNOS more concentrated in hippocampal pyramidal cells than in any other brain area, whereas nNOS is restricted to occasional interneurons. In many brain regions NADPH diaphorase staining reflects NOS catalytic activity. Hippocampal pyramidal cells do not stain for diaphorase with conventional paraformaldehyde fixation but stain robustly with glutaraldehyde fixatives, presumably reflecting eNOS catalytic activity. eNOS in hippocampal pyramidal cells may generate the NO that has been postulated as a retrograde messenger of long-term potentiation.

Modulation of the neuronal omega-conotoxin GVIA-sensitive N-type voltage-dependent calcium channel (VDCC) by neurotransmitters and guanine nucleotides suggests a dynamic interaction between activated G-protein alpha subunits and the N-type VDCC. Our previous report on the purification of the N-type VDCC (McEnery, M. W., Snowman, A. M., Sharp, A. H., Adams, M. E., and Snyder, S. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11095-11099), has led us to investigate a possible association of CTXR with an endogenous G alpha subunit. The addition of the G-protein activator AIF4- modulated the 125I-CTX binding characteristics of the solubilized CTXR. Further immunological analyses employing G alpha subunit-specific antibodies to monitor the cofractionation of G alpha with 125I-CTX binding activity throughout the purification procedure indicate the selective recovery of Go alpha in the purified CTXR preparation, as neither Gs alpha, Gi alpha, nor G beta gamma could be detected. Furthermore, Go alpha associated with CTXR acted as a substrate for pertussis toxin-dependent ADP-ribosylation only upon the addition of exogenous G beta gamma subunits. These results strongly indicate a high affinity complex between an activated Go alpha and CTXR maintained throughout biochemical purification of the 125I-CTX receptor.

The inositol pyrophosphate, diphosphoinositol pentakisphosphate, regulates p53 and protein kinase Akt signaling, and its aberrant increase in cells has been implicated in apoptosis and insulin resistance. Inositol hexakisphosphate kinase-2 (IP6K2), one of the major inositol pyrophosphate synthesizing enzymes, mediates p53-linked apoptotic cell death. Casein kinase-2 (CK2) promotes cell survival and is upregulated in tumors. We show that CK2 mediated cell survival involves IP6K2 destabilization. CK2 physiologically phosphorylates IP6K2 at amino acid residues S347 and S356 contained within a PEST sequence, a consensus site for ubiquitination. HCT116 cells depleted of IP6K2 are resistant to cell death elicited by CK2 inhibitors. CK2 phosphorylation at the degradation motif of IP6K2 enhances its ubiquitination and subsequent degradation. IP6K2 mutants at the CK2 sites that are resistant to CK2 phosphorylation are metabolically stable.

THE immunophilins cyclophilin and FK506 binding protein (FKBP) are small, predominantly soluble proteins that bind the immunosuppressant drugs cyclosporin A and FK506, respectively, with high affinity, and which seem to mediate their pharmacological actions1,2. The Ca2+-dependent protein phosphatase, calcineurin, binds the cyclophilin—cyclosporin A and FKBP—FK506 complexes, indicating that calcineurin might mediate the actions of these drugs3. A physiological role for the immunophilins in the nervous system is implied by a close homology between the structure of NINA A, a protein in the neural retina of Drosophila, and cyclophilin4,5, as well as by the high density of FKBP messenger RNA in brain tissue6. Here we report that the levels of FKBP and mRNA in rat brain are extraordinarily high and that their regional localization is virtually identical to that of calcineurin, indicating that there may be a physiological link between calcineurin and the immunophilins. We also show that at low concentrations FK506 and cyclosporin A enhance the phosphorylation of endogenous protein substrates in brain tissue and in intact PC12 cells, indicating that these drugs may inhibit phosphatase activity by interacting with the immunophilin—calcineurin complexes.

The mitochondrial benzodiazepine receptor (mBzR) has been solubilized with retention of reversible ligand binding, and the associated subunits were characterized. mBzR comprises immunologically distinct protein subunits of 18-, 30-, and 32-kDa. The 18-kDa protein is labeled by the isoquinoline carboxamide mBzR ligand [3H]PK14105, whereas the 30- and 32-kDa subunits are labeled by the benzodiazepine (Bz) ligands [3H]flunitrazepam and [3H]AHN-086. Selective antibodies and reagents identify the 32- and 30-kDa proteins as the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ADC), respectively. While isoquinoline carboxamide and Bz ligands target different subunits, they interact allosterically, as the binding of Bz and isoquinoline carboxamide ligands is mutually competitive at low nanomolar concentrations. Moreover, eosin-5-maleimide and mercuric chloride inhibit [3H]PK11195 binding to the intact receptor via sulfhydryl groups that are present in ADC. VDAC and ADC, outer and inner mitochondrial membrane channel proteins, respectively, together with the 18-kDa subunit, may comprise mBzR at functionally important transport sites at the junction of two mitochondrial membranes.

SummaryThe inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3β and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3β signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.

The omega-conotoxin GVIA (CTX) receptor has been purified 1900-fold to apparent homogeneity by monitoring both reversible binding of 125I-labeled CTX (125I-CTX) and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-125I-CTX (HSA-125I-CTX). Photoincorporation of HSA-125I-CTX into a 230-kDa protein exhibits a pharmacologic and chromatographic profile indicating that the 230-kDa protein is the CTX-binding subunit of the receptor. The pharmacologic specificity of 125I-CTX binding to the purified CTX receptor closely resembles that of the native membrane-bound form with respect to sensitivity towards CTX (Kd = 32 pM) and other peptide toxin antagonists. The purified CTX receptor comprises the 230-kDa protein (alpha 1) and four additional proteins with apparent molecular masses of 140 (alpha 2), 110, 70 (beta 2), and 60 (beta 1) kDa. This subunit structure closely resembles that of the 1,4-dihydropyridine-sensitive L-type calcium channel.

We have characterized the odorant binding properties of purified bovine odorant-binding protein (OBP) using as a ligand [3H]3,7-dimethyloctan-1-ol ([3H]DMO). A broad variety of odorants, including terpenes, aldehydes, esters, and musks, bind to OBP with affinities of 0.2 to 100 microM. Odorant affinities for OBP correlate most closely with their stimulation of an odorant-sensitive adenylyl cyclase as well as hydrophobicity. We also measured the kinetics of binding for the ligands, [3H]DMO and 2-isobutyl-3-[3H]methoxypyrazine. Dissociation of both is markedly accelerated in the presence of excess unlabeled ligand. Competition curves of displacers for [3H]DMO binding are shallow, and saturation binding isotherms for 3H-odorants are curvilinear. These kinetic and equilibrium binding properties suggest that OBP interactions with odorant ligands are negatively cooperative.

In a previous study, we showed that the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP 7 ) physiologically phosphorylates mammalian and yeast proteins. We now report that this phosphate transfer reflects pyrophosphorylation. Thus, proteins must be prephosphorylated by ATP to prime them for IP 7 phosphorylation. IP 7 phosphorylates synthetic phosphopeptides but not if their phosphates have been masked by methylation or pyrophosphorylation. Moreover, IP 7 phosphorylated peptides are more acid-labile and more resistant to phosphatases than ATP phosphorylated peptides, indicating a different type of phosphate bond. Pyrophosphorylation may represent a novel mode of signaling to proteins.

The atypical antipsychotic drugs (AAPDs) have markedly enhanced the treatment of schizophrenias but their use has been hindered by the major weight gain elicited by some AAPDs. We report that orexigenic AAPDs potently and selectively activate hypothalamic AMP-kinase, an action abolished in mice with deletion of histamine H1 receptors. These findings may afford a means of developing more effective therapeutic agents and provide insight into the hypothalamic regulation of food intake.

The inositol pyrophosphates IP 7 and IP 8 contain highly energetic pyrophosphate bonds. Although implicated in various biologic functions, their molecular sites of action have not been clarified. Using radiolabeled IP 7 , we detected phosphorylation of multiple eukaryotic proteins. We also observed phosphorylation of endogenous proteins by endogenous IP 7 in yeast. Phosphorylation by IP 7 is nonenzymatic and may represent a novel intracellular signaling mechanism.

The inositol pyrophosphate disphosphoinositol pentakisphosphate (PP-InsP(3)/InsP(7)) is formed in mammals by two recently cloned inositol hexakiphosphate kinases, InsP(6)K1 and InsP(6)K2 (Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., and Snyder, S. H. (1999) Curr. Biol. 9, 1323-1326). We now report the identification, cloning, and characterization of a third InsP(7) forming enzyme designated InsP(6)K3. InsP(6)K3 displays 50 and 45% sequence identity to InsP(6)K1 and InsP(6)K2, respectively, with a smaller mass (46 kDa) and a more basic character than the other two enzymes. InsP(6)K3 is most enriched in the brain where its localization resembles InsP(6)K1 and InsP(6)K2. Intracellular disposition discriminates the three enzymes with InsP(6)K2 being exclusively nuclear, InsP(6)K3 predominating in the cytoplasm, and InsP(6)K1 displaying comparable nuclear and cytosolic densities.

Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3 ]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-Ins P 4 ) from Ins P 5 . Additionally, mIPMK forms Ins P 4 from Ins(1,4,5) P 3 and Ins P 5 from Ins(1,3,4,5) P 4 .

Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson's disease are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic Parkinson's disease. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide, termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and are investigated by site-directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with Parkinson's disease, suggesting that this loss may be pathologic. This implies that hydrogen sulfide donors may be therapeutic.

The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC 1.14.13.39) has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.

First-generation H 1 -antagonist antihistamines, such as hydroxyzine, have the ability to cross the blood-brain barrier and cause sedation, which limits their usefulness in the treatment of allergic disorders. Cetirizine, a carboxylated metabolite of hydroxyzine, possesses the parent compound's antihistaminic activity but causes less sedation. We compared the activity of cetirizine at central H 1 sites with that of hydroxyzine and terfenadine. We also compared the ability of cetirizine and three antihistamines to cross the blood-brain barrier. In each case we found that the drug's potency at H 1 receptors in the central nervous system was similar to its activity in displacing H 1 receptors in the lung. However, the selectivity for H 1 receptors varied widely from drug to drug. Cetirizine did not bind at any of the receptors investigated, except H 1 sites, even at concentrations as high as 10 μmol/L. Hydroxyzine and dexchlorpheniramine and, to a lesser extent, terfenadine crossed the blood-brain barrier in significant amounts. Cetirizine passed into the central nervous system only half as readily as terfenadine. These findings suggest that cetirizine's low incidence of sedative effects is most likely caused by its diminished potential to cross the blood-brain barrier and also may be partly the result of its greater selectivity for H 1 receptors, compared with its effect at other receptors that may be involved in sedation.

Amiodarone and its pharmacologically active metabolite desethylamiodarone have a sodium channel blocking action that explains some of their antiarrhythmic efficacy. However, the well-documented depression of the calcium channel-dependent sinus node and atrioventricular node function that occurs with amiodarone therapy suggests that amiodarone also blocks calcium influx through voltage-dependent calcium channels. Recent electrophysiologic data support the notion that amiodarone, but not desethylamiodarone, acts as a calcium channel antagonist. In this study, the effects of amiodarone and desethylamiodarone on calcium antagonist receptors associated with the voltage-dependent calcium channels were characterized. Amiodarone, but not its active metabolite desethylamiodarone, was a potent competitor at dihydropyridine and phenylalkylamine (verapamil-like) calcium antagonist binding sites in rat heart, brain, and skeletal and smooth muscles. Substantial inhibition of calcium antagonist binding was retained even after extensive washing of membranes and 2 days after in vivo treatment of rats with amiodarone. The pattern of inhibition of calcium antagonist binding suggests that amiodarone acts at phenylalkylamine binding sites. It is suggested that the acute effects of amiodarone--sinus and atrioventricular node inhibition, vasodilatation, and negative inotropic actions--may reflect calcium antagonist influences of amiodarone itself. Chronic effects of drug therapy, such as inhibition of ventricular conduction by sodium channel blockade, may selectively involve desethylamiodarone.

The high-affinity cannabinoid site in rat brain is an integral component of brain membranes that recognizes cannabinoids with inhibitory constants (Ki) in the nanomolar range. To clarify its physiological role, we studied the regulation of [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) binding. The site is inhibited by heavy metal ions, such as La3+, at low micromolar concentrations; divalent cations, such as Ca2+ and Mg2+, inhibit [3H]TMA binding, though at somewhat higher concentrations. In contrast, [3H]TMA binding is stimulated by Fe2+, Cu2+, and Hg2+ ions. Ascorbic acid and its analogs are also stimulators of cannabinoid binding at low micromolar concentrations. Stimulation of [3H]TMA binding by ascorbate or ions is dependent upon molecular oxygen, but is not inhibited by metabolic poisons. Metabolically stable nucleoside triphosphate analogs enhance [3H]TMA binding by different mechanisms, with hydrolysis of a high-energy phosphate bond apparently requisite for these influences. These results suggest that the cannabinoid binding site is associated with a nucleotide-utilizing protein possessing multiple regulatory subsites.

The cage convulsant [ 35 S]tert-butylbicyclophosphorothionate ([ 35 S]TBPS) labels a presumed sedative-convulsant receptor complex. The relative potencies of barbiturates in competing for [ 35 S]TBPS binding parallels their potencies in enhancing benzodiazepine receptor binding. Barbiturates inhibit [ 35 S]TBPS binding in a complex, mixed competitive fashion, leading to a decrease in both the apparent affinity of TBPS for its sites and the apparent number of TBPS sites. All of the barbiturates examined markedly accelerate the dissociation of [ 35 S]TBPS from its recognition sites, while picrotoxinin does not affect the dissociation. These results suggest that the barbiturate and picrotoxinin/TBPS recognition sites are distinct but allosterically linked.