Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.     

Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [³⁵S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gα_q but have no effect on stimulation of Gα_i or Gα_s. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gα_q and Gα_i but decrease stimulation of Gα_s. Consistent with these functional data, NHERF2 formed cellular complexes with both Gα_q and Gα_i, whereas NHERF1 was found to interact only with Gα_q. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.     

Tankyrase-1 assembly to large protein complexes blocks its telomeric function

Tankyrase-1 poly(ADP-ribosyl)ates the telomere-binding protein TRF1. This post-translational modification dissociates TRF1 from telomeres, enhancing telomerase-mediated telomere elongation. Tankyrase-1 multimerizes via its sterile alpha motif domain, but its functional implication remains elusive. Here, we found that excessive amounts of tankyrase-1 form punctate nuclear foci. This focus formation depends on both homophilic multimerization and heterophilic protein-protein interaction. These foci are functionally dormant because they do not efficiently release TRF1 from telomeres. Consistently, hyper-overexpression of tankyrase-1 attenuates its ability to elongate telomeres. These observations suggest that tankyrase-1 assembly to large protein complexes masks its telomeric function.

Structured summary

MINT-7987689, MINT-7987677: Tankyrase-1 (uniprotkb:O95271) and TRF1 (uniprotkb:P54274) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7987977: Tankyrase-1 (uniprotkb:O95271) physically interacts (MI:0915) with TRF1 (uniprotkb:P54274) by anti tag coimmunoprecipitation (MI:0007)MINT-7987998: Tankyrase-1 (uniprotkb:O95271) physically interacts (MI:0915) with Tankyrase-1 (uniprotkb:O95271) by anti tag coimmunoprecipitation (MI:0007).     

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

TNF regulates cellular NAD+ metabolism in primary macrophages

The inflammatory cytokine TNF is known to affect glucose and lipid metabolism, where its action leads to a cachexic state. Despite a well-established connection of TNF to metabolism, the relationship between TNF and NAD(+) metabolism remains unclear. In this report, we evaluated the effects of TNF on NAD(+) metabolism in cells that are TNF's primary autocrine target-macrophages. We designed real-time PCR primers to all NAD(+) metabolic enzymes, which we used to examine TNF-induced changes over time. We found that TNF paradoxically up-regulated enzymes that served to increase NAD(+) levels, such as IDO and PBEF, as well as enzymes that decrease NAD(+) levels, such as CD38 and CD157. The significance of these mRNA changes was evaluated by examining TNF-mediated changes in cellular NAD(+) levels. Treatment of macrophages with TNF decreased NAD(+) levels over time, suggesting that increases in NAD(+)-degrading enzymes were dominant. To evaluate whether this was the case, we measured TNF-mediated changes in NAD(+) levels in animals where CD38 was genetically deleted. In CD38-/- macrophages, the effects of TNF were reversed, with TNF increasing NAD(+) levels over time. The significance of our findings is threefold: (1) we establish that TNF affects NAD(+) metabolism by regulating the expression of major NAD(+) metabolic enzymes, (2) TNF-induced decreases in cellular NAD(+) levels were carried out through the up-regulation of extracellularly situated enzymes, and (3) we provide a mechanism for the observed clinical connection of TNF-dependent diseases to tissue reductions in NAD(+) content.     

Decreased cADPR and increased NAD+ in the Cd38-/- mouse

CD38 is a type II glycoprotein that catalyzes the formation of cyclic ADP-ribose (cADPR), an intracellular calcium signalling molecule, from nicotinamide adenine dinucleotide (NAD⁺). Using a modified version of the fluorimetric cycling assay for cADPR which reduces between-subject variability, we report significant decreases in brain and lung cADPR, which although similar to previously published values, showed much less individual variation. The reduced variation within each group suggests that the range of cADPR is narrower than previously thought, and that the regulatory mechanisms controlling these levels are more finely tuned. We also report significant increases in brain, lung, and kidney NAD⁺ in the Cd38^−/− mouse, and provide the first experimental demonstration of the proximate relationship between CD38 and NAD⁺.     

Characterization of the Mechanism and Magnitude of Cytoglobin-mediated Nitrite Reduction and Nitric Oxide Generation under Anaerobic Conditions

Cytoglobin (Cygb) is a recently discovered cytoplasmic heme-binding globin. Although multiple hemeproteins have been reported to function as nitrite reductases in mammalian cells, it is unknown whether Cygb can also reduce nitrite to nitric oxide (NO). The mechanism, magnitude, and quantitative importance of Cygb-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, spectrophotometric measurements, and chemiluminescence NO analyzer studies were performed. Under anaerobic conditions, mixing nitrite with ferrous-Cygb triggered NO formation that was trapped and detected using EPR spin trapping. Spectrophotometric studies revealed that nitrite binding to ferrous-Cygb is followed by formation of ferric-Cygb and NO. The kinetics and magnitude of Cygb-mediated NO formation were characterized. It was observed that Cygb-mediated NO generation increased linearly with the increase of nitrite concentration under anaerobic conditions. This Cygb-mediated NO production greatly increased with acidosis and near-anoxia as occur in ischemic conditions. With the addition of nitrite, soluble guanylyl cyclase activation was significantly higher in normal smooth muscle cells compared with Cygb knocked down cells with Cygb accounting for ∼40% of the activation in control cells and ∼60% in cells subjected to hypoxia for 48 h. Overall, these studies show that Cygb-mediated nitrite reduction can play an important role in NO generation and soluble guanylyl cyclase activation under hypoxic conditions, with this process regulated by pH, oxygen tension, nitrite concentration, and the redox state of the cells.     

Tankyrase-1 function at telomeres and during mitosis is regulated by Polo-like kinase-1-mediated phosphorylation

Telomere length is critical for chromosome stability that affects cell proliferation and survival. Telomere elongation by telomerase is inhibited by the telomeric protein, TRF1. Tankyrase-1 (TNKS1) poly(ADP-ribosyl)ates TRF1 and releases TRF1 from telomeres, thereby allowing access of telomerase to the telomeres. TNKS1-mediated poly(ADP-ribosyl)ation also appears to be crucial for regulating the mitotic cell cycle. In searching for proteins that interact with polo-like kinase-1 (Plk1) by using complex proteomics, we identified TNKS1 as a novel Plk1-binding protein. Here, we report that Plk1 forms a complex with TNKS1 in vitro and in vivo, and phosphorylates TNKS1. Phosphorylation of TNKS1 by Plk1 appears to increase TNKS1 stability and telomeric poly(ADP-ribose) polymerase (PARP) activity. By contrast, targeted inhibition of Plk1 or mutation of phosphorylation sites decreased the stability and PARP activity of TNKS1, leading to distort mitotic spindle-pole assembly and telomeric ends. Taken together, our results provide evidence of a novel molecular mechanism in which phosphorylation of TNKS1 by Plk1 may help regulate mitotic spindle assembly and promote telomeric chromatin maintenance.     

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A_2A receptor (A_2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α_olf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ₇ subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ₇ protein controls the hierarchial assembly of a specific G-protein α_olfβ₂γ₇ heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A_2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ₇ subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A_2AR G-protein α_olfβ₂γ₇ interface as a possible therapeutic target for Parkinson disease.     

A Single Residue in a Novel ADP-ribosyl Cyclase Controls Production of the Calcium-mobilizing Messengers Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate are ubiquitous calcium-mobilizing messengers produced by the same family of multifunctional enzymes, the ADP-ribosyl cyclases. Not all ADP-ribosyl cyclases have been identified, and how production of different messengers is achieved is incompletely understood. Here, we report the cloning and characterization of a novel ADP-ribosyl cyclase (SpARC4) from the sea urchin, a key model organism for the study of calcium-signaling pathways. Like several other members of the ADP-ribosyl cyclase superfamily, SpARC4 is a glycoprotein targeted to the plasma membrane via a glycosylphosphatidylinositol anchor. However, unlike most other members, SpARC4 shows a remarkable preference for producing cyclic ADP-ribose over nicotinic acid adenine dinucleotide phosphate. Mutation of a single residue (tyrosine 142) within a noncanonical active site reversed this striking preference. Our data highlight further diversification of this unusual enzyme family, provide mechanistic insight into multifunctionality, and suggest that different ADP-ribosyl cyclases are fine-tuned to produce specific calcium-mobilizing messengers.