Science's STKE

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ISSN / EISSN : 1525-8882 / 1525-8882
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Nancy R. Gough
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007tw458

Abstract:
Previous studies with knockout mice implicated the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases in regulation of innate immune responses. Rothlin et al. characterized the function of the receptor family in dendritic cells and showed that they stimulate the production of SOCS (suppressor of cytokine signaling) proteins, which are inhibitors of Toll-like receptor (TLR) signaling, thereby contributing to termination of the innate immune response. Splenic dendritic cells from single-, double-, or triple-knockout mice showed progressively severe hyperresponsiveness to stimuli that activate TLR3, TLR4, or TLR9, suggesting that the TAM family members are important inhibitors of multiple TLR pathways. In isolated mouse dendritic cells, only Mer and Axl were detected, and overnight activation of these cells with the TAM ligands Gas6 or ProS inhibited cytokine production stimulated by activation of TLR3, TLR4, or TLR9. The inhibition of TLR signaling by activation of the TAM proteins required new gene expression and protein synthesis, and screening for likely TLR inhibitors revealed that both the genes encoding SOCS1 and SOCS3 were stimulated in response to exposure of dendritic cells to Gas6. Gas6 stimulated the phosphorylation of the transcription factor STAT1, and STAT1 was required for TAM-mediated activation of SOCS1 and SOCS3 gene expression. TAM-mediated activation of STAT1 required the type I interferon (IFN) receptor subunit INFAR1, because Gas6 failed to stimulate SOCS1 expression in dendritic cells from infar1 knockout mice. A TAM ligand-dependent interaction was detected between Axl and INFAR1 (by coimmunoprecipitation assay). Abundance of Axl mRNA was increased after TLR activation through a mechanism requiring STAT1 and INFAR1. IFN-mediated induction of SOCS1 expression was substantially lost in TAM knockout dendritic cells. Coapplication of IFNα and Gas6 to dendritic cells increased SOCS1 mRNA production more than either ligand alone; however, expression of cytokine signaling stimulators by IFNα was inhibited by coapplication of Gas6. The authors suggest that there is a three-step inflammatory cycle: (i) activation of TLR signaling by a pathogen, (ii) amplification of the signal through production of IFN, and (iii) increased synthesis of the TAM proteins and production of SOCS proteins to inhibit TLR signaling. See O'Neill for commentary. C. V. Rothlin, S. Ghosh, E. I. Zuniga, M. B. A. Oldstone, G. Lemke, TAM receptors are pleiotropic inhibitors of the innate immune response. Cell 131, 1124-1136 (2007). [PubMed] L. A. J. O'Neill, TAMpering with Toll-like receptor signaling. Cell 131, 1039-1041 (2007). [PubMed]
L. Bryan Ray, Pamela J. Hines
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007tw460

Abstract:
Circadian rhythms in plants and animals appear to be coupled to periodic changes in activity of metabolic pathways (see the Perspective by Imaizumi et al.). Yin et al. describe a molecular mechanism that may contribute to the coordination of these biochemical processes. Rev-erbα controls transcription of the gene encoding the circadian clock component Bmal1. Rev-erbα binds to and is regulated by heme, which stabilizes Rev-erbα in a repressor complex, which in turn can block production of gluconeogenic enzymes. Thus, Rev-erbα acts as a heme sensor to coordinate the cellular clock, glucose homeostasis, and energy metabolism in human liver cells. Studying Arabidopsis, Dodd et al. now show that a cytoplasmic signaling molecule, cyclic adenosine diphosphate ribose (cADPR), is also a component of the clock mechanism. Perturbations to the feedback loop including cADPR result in instabilities in the clock and disruptions in the daily oscillations of cytoplasmic Ca2+ release. L. Yin, N. Wu, J. C. Curtin, M. Qatanani, N. R. Szwergold, R. A. Reid, G. M. Waitt, D. J. Parks, K. H. Pearce, G. B. Wisely, M. A. Lazar, Rev-erbα, a heme sensor that coordinates metabolic and circadian pathways. Science 318, 1786-1789 (2007). [Abstract] [Full Text] A. N. Dodd, M. J. Gardner, C. T. Hotta, K. E. Hubbard, N. Dalchau, J. Love, J.-M. Assie, F. C. Robertson, M. K. Jakobsen, J. Gonçalves, D. Sanders, A. A. R. Webb, The Arabidopsis circadian clock incorporates a cADPR-based feedback loop. Science 318, 1789-1792 (2007). [Abstract] [Full Text] T. Imaizumi, S. A. Kay, J. I. Schroeder, Daily watch on metabolism. Science 318, 1730-1731 (2007). [Summary] [Full Text]
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007pe73

Abstract:
Toll–interleukin-1 receptor (TIR) domain–containing proteins are best known as critical players in vertebrate immune defense against pathogens. Four of the five members of this family are required for the activation of immune cells downstream of the engagement of Toll-like receptors (TLRs) by microbial molecules. Mice deficient in any one of these four molecules show greatly enhanced susceptibility to specific classes of pathogens. However, the physiological function of the fifth mammalian protein, sterile alpha and TIR motif–containing 1 [SARM1, also known as myeloid differentiation marker 88-5 (MyD88-5)], has remained elusive. Now, the study of the SARM1 reporter and SARM1-deficient mice has uncovered an unanticipated function of this molecule in the regulation of neuronal survival in response to metabolic stress. Together with pioneering observations on the functions of TIR-1, the worm ortholog of SARM1, and other reports on the role of TLRs in neuronal development and responses to injury in mammals, these exciting results suggest that further studies of SARM1-deficient animals may uncover unexpected similarities between the ways in which neurons and immune cells sense and respond to danger.
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007pl7

Abstract:
Membrane domains, such as caveolae and clathrin-coated pits, regulate cell signaling and protein internalization in the plasma membrane. Fluorescence imaging and microscopy provide an opportunity to determine the receptor protein dynamics of membrane microdomains. The family of image correlation spectroscopy (ICS) techniques provides powerful tools with which to measure the aggregation, clustering, and dynamics of proteins in the plasma membrane. ICS is used to calculate the cluster density and the degree of aggregation of plasma membrane proteins, whereas image cross-correlation spectroscopy (ICCS) measures the fraction of colocalization of two proteins. Dynamic image correlation spectroscopy (DICS) can be used to analyze protein dynamics on the cell surface during live-cell imaging.
Nancy R. Gough
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007tw461

Abstract:
Chronic lung disease, including cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), is characterized by high concentrations of interleukin-8 (IL-8) in cells around the airway and accumulation of neutrophils, yet the lungs of these patients are frequently colonized by bacteria, such as Pseudomonas aeruginosa. Hartl et al. show that IL-8 acts through the CXCR1 receptor, not the CXCR2 receptor, to promote the bactericidal activity of neutrophils. By comparing neutrophils from the peripheral blood and airway (bronchial alveolar lavage and sputum) of healthy individuals with those from patients with cystic fibrosis, bronchiectasis, or COPD, Hartl et al. found that the airway neutrophils from the diseased lungs had decreased CXCR1 at the surface and exhibited impaired bactericidal activity. The protease elastase is increased in abundance in airway fluids from patients with cystic fibrosis. Further statistical analysis of various factors associated with cystic fibrosis, including infection with P. aeruginosa, suggested that the concentration of free elastase correlated with the loss of CXCR1 on the airway neutrophils. Addition of airway fluids from individuals with cystic fibrosis to peripheral neutrophils caused loss of CXCR1 and impaired bacterial killing, and these effects were prevented if the elastase activity was inhibited pharmacologically in the airway fluid prior to addition to the isolated neutrophils. Cleavage of CXCR1 by elastase produced two soluble CXCR1 fragments that were also detected in the airway fluids from patients with cystic fibrosis, bronchiectasis, or COPD. Application of purified soluble CXCR1 fragments stimulated bronchial epithelial cells to produce IL-8 through a mechanism requiring the Toll-like receptor TLR2. Cystic fibrosis patients who inhaled α1 antitrypsin to inhibit elastase activity for 4 weeks showed increased CXCR1 at the surface of airway neutrophils, decreased abundance of soluble CXCR1 fragments in the airway fluid, and decreased P. aeruginosa in the sputum. In commentary, Sabroe and Whyte describe how failure of mucosal immunity contributes to disease pathogenesis, especially in cystic fibrosis. D. Hartl, P. Latzin, P. Hordijk, V. Marcos, C. Rudolph, M. Woischnik, S. Krauss-Etschmann, B. Koller, D. Reinhardt, A. A. Roscher, D. Roos, M. Griese, Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat. Med. 13, 1423-1430 (2007). [PubMed] I. Sabroe, M. K. B. Whyte, Incapacitating the immune system in cystic fibrosis. Nat. Med. 13, 1417-1418 (2007). [PubMed]
Valda Vinson
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007tw457

Abstract:
Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that can initiate a variety of signaling events. Many human cancers involve mutations that activate PI3Kα, a heterodimer composed of a catalytic subunit, p110α, and a regulatory subunit, p85α, both of which contain multiple domains. Huang et al. describe the crystal structure of a complex between the full-length human p110α catalytic subunit and the binding and activation domains of the p85α regulatory subunit. The structure provides insight into how oncogenic mutations affect enzyme activity and could assist in the future design of isoform- or mutation-specific inhibitors. C.-H. Huang, D. Mandelker, O. Schmidt-Kittler, Y. Samuels, V. E. Velculescu, K. W. Kinzler, B. Vogelstein, S. B. Gabelli, L. M. Amzel, The structure of a human p110α/p85α complex elucidates the effects of oncogenic PI3Kα mutations. Science 318, 1744-1748 (2007). [Abstract] [Full Text]
Elizabeth M. Adler
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007tw456

Abstract:
Polarization of various cell types depends on a complex containing the PDZ domain-containing scaffolding proteins Par-3 and Par-6 and atypical protein kinase C. Noting that phosphoinositides (PIPs) have also been implicated in cell polarization and that PDZ domains--although better known for mediating protein-protein interactions--can bind phosphatidylinositol (PI) lipids, Wu et al. investigated interactions between Par protein PDZ domains and PI lipids. The second Par-3 PDZ domain (PDZ2) bound to brain-derived liposomes with an affinity comparable to that of the PLCδ pleckstrin homology (PH) domain (a PI lipid-binding domain); strip-based assays and experiments with defined liposomes indicated that it bound directly to PIPs. Lipid-binding assays of 74 human PDZ domains and 14 PDZ tandems revealed that a subset bound PI lipids. Structural and biochemical analysis indicated that lipid binding by the Par-3 PDZ2 depended on a cluster of positively charged residues and that PDZ2 contained a PIP head group-binding pocket that overlapped its peptide ligand-binding site. In MDCK epithelial cells, Par-3 primarily localized to the cell membrane; mutational analysis indicated that membrane localization depended on PI lipid binding by PDZ2. Par-3 knockdown disrupted tight junction formation and reestablishment of polarization in MDCK cells. Epithelial repolarization was rescued by wild-type Par-3 or by a mutant in which the PLCδ PH domain replaced PDZ2 but not by mutants defective in lipid binding. The Par-3 PDZ3 domain bound to the lipid phosphatase PTEN. The authors conclude that lipid binding by the Par-3 PDZ2 domain is critical to its role in epithelial polarization and that the combination of PIP-binding by PDZ2 and PTEN-binding by PDZ3 could enable Par-3 to integrate PIP signaling. H. Wu, W. Feng, J. Chen, L.-N. Chan, S. Huang, M. Zhang, PDZ domains of Par-3 as potential phosphoinositide signaling integrators. Mol. Cell 28, 886-898 (2007). [PubMed]
John F. Foley
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007tw455

Abstract:
Motile cilia are plasma membrane projections that contain a central core of microtubules whose movement relative to each other contributes to ciliary beating. The left-right body patterning of developing embryos is thought to result from cilia-dependent differential distribution of morphogens. Knockout of inositol 1,3,4,5,6-pentakisphosphate (IP5) 2-kinase (Ipk1), the enzyme that synthesizes IP6, in zebrafish embryos results in defective left-right positioning of organs, although the mechanism involved is unknown. Sarmah et al. extended this work and knocked down expression of ipk1 in zebrafish by injecting embryos with an antisense morpholino oligonucleotide (MO) targeted to ipk1 mRNA (ipk1MO1 embryos). The Kupffer’s vesicle (KV) is the organ that initiates left-right organ placement in the zebrafish. The authors found that whereas KV cilia from wild-type embryos beat with a regular frequency in a counterclockwise direction, those from ipk1MO1 embryos vibrated without a defined direction of motion. Furthermore, immunocytochemical analyses revealed that cilia in ipk1MO1 embryos were shorter than those in wild-type embryos. Although the ectopic expression in ipk1MO1 embryos of Ipk1 resulted in the rescue of ciliary length and motion, that of a catalytically inactive mutant of Ipk1 did not. Fluorescence microscopy showed that Ipk1 was localized to centrosomes in the basal bodies of cilia in wild-type embryos. Transmission electron microscopy demonstrated that the organization of microtubules in the cilia from ipk1MO1 embryos was similar to that in wild-type embryos. However, the anterograde microtubule-dependent movement of melanosomes (pigmented vesicles), which is mediated by kinesin proteins, was much slower in ipk1MO1 cilia than in wild-type cilia. Together these data implicate Ipk1, and potentially its product IP6, in the maintenance of cilia length and the regulation of ciliary motion. B. Sarmah, V. P. Winfrey, G. E. Olson, B. Appel, S. R. Wente, A role for the inositol kinase Ipk1 in ciliary beating and length maintenance. Proc. Natl. Acad. Sci. U.S.A. 104, 19843-19848 (2007). [Abstract] [Full Text]
L. Bryan Ray
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007tw459

Abstract:
Hirayama et al. provide new understanding of the signaling feedback loops that control the mammalian circadian clock. CLOCK and BMAL1 are clock proteins that form a complex and promote clock-controlled transcription of target genes. CLOCK is a histone acetyltransferase that acts to regulate gene expression by initiating remodeling of chromatin. The new work showed that CLOCK acetylated its binding partner BMAL1 when transfected, tagged proteins were expressed in cultured cells or when purified proteins were mixed in vitro. Reconstitution of mouse embryo fibroblasts lacking BMAL1 with a BMAL mutant that lacked the lysine residue acetylated by CLOCK failed to restore circadian gene regulation. Acetylation of BMAL1 appeared to increase association of the CLOCK-BMAL1 complex with cryptochrome 1 (Cry1), which acts as a repressor of CLOCK-BMAL1-induced transcription. Cry1-mediated repression was lacking in cells expressing the acetylation-deficient mutant of BMAL1, and interaction of Cry1 with the CLOCK-BMAL1 complex was lost in a two-hybrid assay with the mutant BMAL1. Acetylation of BMAL1 in vivo also occurs at a time in the circadian cycle when transcription of clock-controlled genes is diminished. Thus, the authors conclude that acetylation of BMAL1 by its binding partner CLOCK contributes to a negative feedback loop that decreases clock-regulated transcription. The positive and negative feedback loops thus appear to be tightly associated because CLOCK’s effect on BMAL contrasts with its role in acetylation of histones, which stimulates transcription of clock-dependent genes. J. Hirayama, S. Sahar, B. Grimaldi, T. Tamaru, K. Takamatsu, Y. Nakahata, P. Sassone-Corsi, CLOCK-mediated acetylation of BMAL1 controls circadian function. Nature, 450, 1086-1090 (2007). [PubMed]
Ulla Schwertassek, Lars Weingarten, Tobias P Dick
Science's STKE, Volume 2007; https://doi.org/10.1126/stke.4172007pl8

Abstract:
A number of thiol-dependent oxidoreductases are released from cells and act on the cell surface. Correspondingly, several cell-surface processes appear to depend on catalyzed thiol-disulfide exchange, including integrin activation and the fusion of viral particles with the host membrane. Tumor cells frequently increase the abundance of secreted and cell-surface forms of particular oxidoreductases, and evidence suggests that oxidoreductases released from tumor cells promote growth and contribute to the remodeling of the cellular microenvironment. Few cell-surface or membrane proteins that are targeted by extracellular redox enzymes have been identified. One major reason for this slow progress is the highly transient nature of thiol-disulfide exchange, making its detection by conventional techniques difficult or impossible. Here we describe the application of an activity-based proteomics approach, also known as "mechanism-based kinetic trapping," to identify individual cell-surface target proteins that engage in disulfide exchange with thiol-dependent oxidoreductases. Although we have applied this approach to thioredoxin-1, it should also be applicable to other members of the thioredoxin superfamily whose activity is based on the CXXC active-site motif.
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