Ubiquitination of Neuronal Nitric-oxide Synthase in the Calmodulin-binding Site Triggers Proteasomal Degradation of the Protein

Nitric-oxide synthase, a cytochrome P450-like hemoprotein enzyme, catalyzes the synthesis of nitric oxide, a critical signaling molecule in a variety of physiological processes. Our laboratory has discovered that certain drugs suicide-inactivate neuronal nitric-oxide synthase (nNOS) and lead to the preferential ubiquitination of the inactivated nNOS by an Hsp70- and CHIP (C terminus of Hsc70-interacting protein)-dependent process. To further understand the process by which altered nNOS is recognized, ubiquitinated, and proteasomally degraded, we examined the sites of ubiquitination on nNOS. We utilized an in vitro ubiquitination system containing purified E1, E2 (UbcH5a), Hsp70, and CHIP that recapitulates the ability of the cells to selectively recognize and ubiquitinate the altered forms of nNOS. LC-MS/MS analysis of the tryptic peptides obtained from the in vitro ubiquitinated nNOS identified 12 ubiquitination sites. Nine of the sites were within the oxygenase domain and two were in the calmodulin-binding site, which links the oxygenase and reductase domains, and one site was in the reductase domain. Mutational analysis of the lysines in the calmodulin-binding site revealed that Lys-739 is a major site for poly-ubiquitination of nNOS in vitro and regulates, in large part, the CHIP-dependent degradation of nNOS in HEK293 cells, as well as in in vitro studies with fraction II. Elucidating the exact site of ubiquitination is an important step in understanding how chaperones recognize and trigger degradation of nNOS.     

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding

Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.     

Glutathione s-transferase omega 1 activity is sufficient to suppress neurodegeneration in a Drosophila model of Parkinson disease

A loss-of-function mutation in the gene parkin causes a common neurodegenerative disease that may be caused by mitochondrial dysfunction. Glutathione S-transferase Omega (GSTO) is involved in cell defense mechanisms, but little is known about the role of GSTO in the progression of Parkinson disease. Here, we report that restoration of Drosophila GSTO1 (DmGSTO1), which is down-regulated in parkin mutants, alleviates some of the parkin pathogenic phenotypes and that the loss of DmGSTO1 function enhances parkin mutant phenotypes. We further identified the ATP synthase β subunit as a novel in vivo target of DmGSTO1. We found that glutathionylation of the ATP synthase β subunit is rescued by DmGSTO1 and that the expression of DmGSTO1 partially restores the activity and assembly of the mitochondrial F(1)F(0)-ATP synthase in parkin mutants. Our results suggest a novel mechanism for the protective role of DmGSTO1 in parkin mutants, through the regulation of ATP synthase activity, and provide insight into potential therapies for Parkinson disease neurodegeneration.     

Evidence for irreversible inhibition of glycogen synthase kinase-3β by tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.     

Regulation of Endothelial Nitric-oxide Synthase (NOS) S-Glutathionylation by Neuronal NOS: EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN MYOCARDIAL CONSTITUTIVE NOS ISOFORMS*

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β₃-adrenergic receptor (β₃-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β₃-AR signaling and found that the β₃-AR-mediated reduction in cell shortening and [Ca²⁺]_i transient amplitude was abolished both in eNOS^−/− and nNOS^−/− left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-l-thiocitrulline. LV superoxide (O₂^˙̄) production was increased in nNOS^−/− mice and reduced by l-N^ω-nitroarginine methyl ester (l-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS^−/− myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O₂^˙̄, only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca²⁺]_i transient response to β₃-AR stimulation in nNOS^−/− mice. In summary, our data show that increased O₂^˙̄ production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β₃-AR stimulation in nNOS^−/− myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS^−/− mice result from disruption of eNOS signaling.     

Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.     

LETM Proteins Play a Role in the Accumulation of Mitochondrially Encoded Proteins in Arabidopsis thaliana and AtLETM2 Displays Parent of Origin Effects

The Arabidopsis thaliana genome contains two genes with homology to the mitochondrial protein LETM1 (leucine zipper-EF-hand-containing transmembrane protein). Inactivation of both genes, Atletm1 and Atletm2, together is lethal. Plants that are hemizygous for AtLETM2 and homozygous for Atletm1 (letm1(−/−) LETM2(+/−)) displayed a mild retarded growth phenotype during early seedling growth. It was shown that accumulation of mitochondrial proteins was reduced in hemizygous (letm1(−/−) LETM2(+/−)) plants. Examination of respiratory chain proteins by Western blotting, blue native PAGE, and enzymatic activity assays revealed that the steady state level of ATP synthase was reduced in abundance, whereas the steady state levels of other respiratory chain proteins remained unchanged. The absence of a functional maternal AtLETM2 allele in an Atletm1 mutant background resulted in early seed abortion. Reciprocal crosses revealed that maternally, but not paternally, derived AtLETM2 was absolutely required for seed development. This requirement for a functional maternal allele of AtLETM2 was confirmed using direct sequencing of reciprocal crosses of Col-0 and Ler accessions. Furthermore, AtLETM2 promoter β-glucuronidase constructs displayed exclusive maternal expression patterns.     

New insights into the organisation and intracellular localisation of the two subunits of glucose-6-phosphatase

Glucose-6 phosphatase (G6Pase), a key enzyme of glucose homeostasis, catalyses the hydrolysis of glucose-6 phosphate (G6P) to glucose and inorganic phosphate. A deficiency in G6Pase activity causes type 1 glycogen storage disease (GSD-1), mainly characterised by hypoglycaemia. Genetic analyses of the two forms of this rare disease have shown that the G6Pase system consists of two proteins, a catalytic subunit (G6PC) responsible for GSD-1a, and a G6P translocase (G6PT), responsible for GSD-1b. However, since their identification, few investigations concerning their structural relationship have been made. In this study, we investigated the localisation and membrane organisation of the G6Pase complex. To this aim, we developed chimera proteins by adding a fluorescent protein to the C-terminal ends of both subunits. The G6PC and G6PT fluorescent chimeras were both addressed to perinuclear membranes as previously suggested, but also to vesicles throughout the cytoplasm. We demonstrated that both proteins strongly colocalised in perinuclear membranes. Then, we studied G6PT organisation in the membrane. We highlighted FRET between the labelled C and N termini of G6PT. The intramolecular FRET of this G6PT chimera was 27%. The coexpression of unlabelled G6PC did not modify this FRET intensity. Finally, the chimera constructs generated in this work enabled us for the first time to analyze the relationship between GSD-1 mutations and the intracellular localisation of both G6Pase subunits. We showed that GSD1 mutations did neither alter the G6PC or G6PT chimera localisation, nor the interaction between G6PT termini. In conclusion, our results provide novel information on the intracellular distribution and organisation of the G6Pase complex.     

Ebselen inhibits iron-induced tau phosphorylation by attenuating DMT1 up-regulation and cellular iron uptake

Dysregulation of iron homeostasis is involved in the pathological process of Alzheimer's disease (AD). We have recently reported that divalent metal transporter 1 (DMT1) is upregulated in an AD transgenic mouse brain, and that silencing of DMT1, which reduces cellular iron influx, results in inhibition of amyloidogenesis in vitro, suggesting a potential target of DMT1 for AD therapy. In the present study, we tested the hypothesis that inhibition of DMT1 with ebselen, a DMT1 transport inhibitor, could affect tau phosphorylation. Human neuroblastoma SH-SY5Y cells were pre-treated with ebselen and then treated with ferrous sulfate (dissolved in ascorbic acid), and the effects of ebselen on tau phosphorylation and the relative signaling pathways were examined. Our results showed that ebselen decreased iron influx, reduced iron-induced ROS production, inhibited the activities of cyclin-dependent kinase 5 and glycogen synthase kinase 3β, and ultimately attenuated the levels of tau phosphorylation at the sites of Thr205, Ser396 and Thr231. The present study indicates that the neuroprotective effect of ebselen on AD is not only related to its antioxidant activity as reported previously, but is also associated with a reduction in tau phosphorylation by inhibition of DMT1.