TrkB receptor signaling in the nucleus tractus solitarius mediates the food intake-suppressive effects of hindbrain BDNF and leptin

Brain-derived neurotrophic factor (BDNF) and TrkB receptor signaling contribute to the central nervous system (CNS) control of energy balance. The role of hindbrain BDNF/TrkB receptor signaling in energy balance regulation is examined here. Hindbrain ventricular BDNF suppressed body weight through reductions in overall food intake and meal size and by increasing core temperature. To localize the neurons mediating the energy balance effects of hindbrain ventricle-delivered BDNF, ventricle subthreshold doses were delivered directly to medial nucleus tractus solitarius (mNTS). mNTS BDNF administration reduced food intake significantly, and this effect was blocked by preadministration of a highly selective TrkB receptor antagonist {[N2-2-2-Oxoazepan-3-yl amino]carbonyl phenyl benzo (b)thiophene-2-carboxamide (ANA-12)}, suggesting that TrkB receptor activation mediates hindbrain BDNF's effect on food intake. Because both BDNF and leptin interact with melanocortin signaling to reduce food intake, we also examined whether the intake inhibitory effects of hindbrain leptin involve hindbrain-specific BDNF/TrkB activation. BDNF protein content within the dorsal vagal complex of the hindbrain was increased significantly by hindbrain leptin delivery. To assess if BDNF/TrkB receptor signaling acts downstream of leptin signaling in the control of energy balance, leptin and ANA-12 were coadministered into the mNTS. Administration of the TrkB receptor antagonist attenuated the intake-suppressive effects of leptin, suggesting that mNTS TrkB receptor activation contributes to the mediation of the anorexigenic effects of hindbrain leptin. Collectively, these results indicate that TrkB-mediated signaling in the mNTS negatively regulates food intake and, in part, the intake inhibitory effects of leptin administered into the NTS.     

The Anx7(+/-) knockout mutation alters electrical and secretory responses to Ca(2+)-mobilizing agents in pancreatic β-cells

Insulin secretion from the pancreatic β-cell is controlled by changes in membrane potential and intracellular Ca(2+). The contribution of intracellular Ca(2+) stores to this process is poorly understood. We have previously shown that β-cells of mice lacking one copy of the Annexin 7 gene (Anx7(+/-)) express reduced levels of IP(3) receptors and defects in IP(3)-dependent Ca(2+) signaling. To further elucidate the effect of the Anx7(+/-) mutation on signaling related to intracellular Ca(2+) stores in the β-cell, we measured the effects of Ca(2+) mobilizing agents on electrical activity, intracellular Ca(2+) and insulin secretion in control and mutant β-cells. We found that the muscarinic agonist carbachol and the ryanodine receptor agonists caffeine and 4-chloro-m-cresol had more potent depolarizing effects on Anx7(+/-) β-cells compared to controls. Accordingly, glucose-induced insulin secretion was augmented to a greater extent by caffeine in mutant islets. Surprisingly, ryanodine receptor-mediated Ca(2+) mobilization was not affected by the Anx7(+/-) mutation, suggesting that the mechanism underlying the observed differences in electrical and secretory responsiveness does not involve intracellular Ca(2+) stores. Our results provide evidence that both IP3 receptors and ryanodine receptors play important roles in regulating β-cell membrane potential and insulin secretion, and that the Anx7(+/-) mutation is associated with alterations in the signaling pathways related to these receptors.     

TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling

Stress-activated mitogen-activated protein (MAP) kinase p38 mediates stress signaling in mammalian cells via threonine and tyrosine phosphorylation in its conserved TGY motif by upstream MAP kinase kinases (MKKs). In addition, p38 MAP kinase can also be activated by an MKK-independent mechanism involving TAB-1 (TAK-1-binding protein)-mediated autophosphorylation. Although TAB-1-mediated p38 activation has been implicated in ischemic heart, the biological consequences and downstream signaling of TAB-1-mediated p38 activation in cardiomyocytes is largely unknown. We show here that TAB-1 expression leads to a significant induction of p38 autophosphorylation and consequent kinase activation in cultured neonatal cardiomyocytes. In contrast to MKK3-induced p38 kinase downstream effects, TAB-1-induced p38 kinase activation does not induce expression of pro-inflammatory genes, cardiac marker gene expression, or changes in cellular morphology. Rather, TAB-1 binds to p38 and prevents p38 nuclear localization. Furthermore, TAB-1 disrupts p38 interaction with MKK3 and redirects p38 localization in the cytosol. Consequently, TAB-1 expression antagonizes the downstream activity of p38 kinase induced by MKK3 and attenuates interleukin-1beta-induced inflammatory gene induction in cardiomyocytes. These data suggest that TAB-1 can mediate MKK-independent p38 kinase activation while negatively modulating MKK-dependent p38 function. Our study not only redefines the functional role of TAB-1 in p38 kinase-mediated signaling pathways but also provides the first evidence that intracellular localization of p38 kinase and complex interaction dictates its downstream effects. These results suggest a previously unknown mechanism for stress-MAP kinase regulation in mammalian cells.     

The membrane-proximal region of the thrombopoietin receptor confers its high surface expression by JAK2-dependent and -independent mechanisms

Janus tyrosine kinase 2 (JAK2) is essential for signaling by the thrombopoietin (TpoR) and erythropoietin (EpoR) receptors. In the absence of JAK2 most EpoR molecules are retained in the endoplasmic reticulum in an Endo H-sensitive form. In contrast, we show that in the absence of JAK2 a large fraction of the TpoR is processed to the mature Endo H-resistant form and reaches the cell surface. By studying chimeras of the TpoR and EpoR we show that high surface expression of the TpoR is entirely conferred by the membrane-proximal region of the intracellular domain that includes the juxtamembrane, Box 1, and Box 2 regions. The TpoR intracellular domain shows similar effects on receptor endocytosis rate as that of the EpoR, but does stabilize the mature receptor isoform from degradation. Co-expression of JAK2 further stabilizes mature TpoR and thus further increases its surface expression. This JAK2 effect depends on the Box 1 region, the only JAK2 interacting site in the TpoR. By contrast, EpoR requires Box 1 as well as the flanking 20 residues on the C-terminal side for JAK2 interaction and JAK2-dependent surface expression. Our study suggests that whereas cell surface expression of type I cytokine receptors requires their cognate JAKs, the mechanisms governing receptor-JAK interactions differ among receptors interacting with the same JAK protein.     

Steady-state kinetics and inhibitory action of antitubercular phenothiazines on mycobacterium tuberculosis type-II NADH-menaquinone oxidoreductase (NDH-2)

Type-II NADH-menaquinone oxidoreductase (NDH-2) is an essential respiratory enzyme of the pathogenic bacterium Mycobacterium tuberculosis (Mtb) that plays a pivotal role in its growth. In the present study, we expressed and purified highly active Mtb NDH-2 using a Mycobacterium smegmatis expression system, and the steady-state kinetics and inhibitory actions of phenothiazines were characterized. Purified NDH-2 contains a non-covalently bound flavin adenine dinucleotide cofactor and oxidizes NADH with quinones but does not react with either NADPH or oxygen. Ubiquinone-2 (Q2) and decylubiquinone showed high electron-accepting activity, and the steady-state kinetics and the NADH-Q2 oxidoreductase reaction were found to operate by a ping-pong reaction mechanism. Phenothiazine analogues, trifluoperazine, Compound 1, and Compound 2 inhibit the NADH-Q2 reductase activity with IC50 = 12, 11, and 13 microm, respectively. Trifluoperazine inhibition is non-competitive for NADH, whereas the inhibition kinetics is found to be uncompetitive in terms of Q2.     

Structure of the Escherichia coli DNA polymerase III epsilon-HOT proofreading complex

The epsilon subunit of Escherichia coli DNA polymerase III possesses 3'-exonucleolytic proofreading activity. Within the Pol III core, epsilon is tightly bound between the alpha subunit (DNA polymerase) and subunit. Here, we present the crystal structure of epsilon in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 A resolution. The epsilon-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the epsilon central beta-sheet as well as interactions between HOT and the catalytically important helix alpha1-loop-helix alpha2 motif of epsilon. This structure provides insight into how HOT and, by implication, may stabilize the epsilon subunit, thus promoting efficient proofreading during chromosomal replication.     

The Fe-CO bond energy in myoglobin: a QM/MM study of the effect of tertiary structure

The Fe-CO bond dissociation energy (BDE) in myoglobin (Mb) has been calculated with B3LYP quantum mechanics/molecular mechanics methods for 22 different Mb conformations, generated from molecular dynamics simulations. Our average BDE of 8.1 kcal/mol agrees well with experiment and shows that Mb weakens the Fe-CO bond by 5.8 kcal/mol; the calculations provide detailed atomistic insight into the origin of this effect. BDEs for Mb conformations with the R carbonmonoxy tertiary structure are on average 2.6 kcal/mol larger than those with the T deoxy tertiary structure, suggesting two functionally distinct allosteric states. This allostery is partly explained by the reduction in distal cavity steric crowding as Mb moves from its T to R tertiary structure.