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  • Synthetic lethality with cisplatin
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-21
    Leslie K. Ferrarelli

    Blocking the kinase MAST1 with lestaurtinib prevents cisplatin resistance in cancers driven by Raf-MEK signaling.

    更新日期:2018-08-22
  • IL-1 plays backup
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-21
    John F. Foley

    Signaling by the cytokine IL-1 provides a backup mechanism to maintain barrier defense during infection by immune-evasive viruses.

    更新日期:2018-08-22
  • Kinase domain dimerization drives RIPK3-dependent necroptosis
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-21
    Saravanan Raju, Daniel M. Whalen, Meron Mengistu, Carter Swanson, John G. Quinn, Susan S. Taylor, Joshua D. Webster, Kim Newton, Andrey S. Shaw

    Necroptosis, an inflammatory form of cell death, is initiated by the activation of receptor-interacting protein kinase 3 (RIPK3), which depends on its interaction with RIPK1. Although catalytically inactive, the RIPK3 mutant D161N still stimulates RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice. Whereas the absence of RIPK1 rescues RIPK3 D161N homozygous mice, we report that the absence of RIPK1 leads to embryonic lethality in RIPK3 D161N heterozygous mice. This suggested that the kinase domain of RIPK3 had a noncatalytic function that was enhanced by a conformation induced by the D161N mutation. We found that the RIPK3 kinase domain homodimerized through a surface that is structurally similar to that of the RAF family members. Mutation of residues at the dimer interface impaired dimerization and necroptosis. Kinase domain dimerization stimulated the activation of RIPK3 through cis-autophosphorylation. This noncatalytic, allosteric activity was enhanced by certain kinase-deficient mutants of RIPK3, including D161N. Furthermore, apoptosis induced by certain RIPK3 inhibitors was also dependent on the kinase dimerization interface. Our studies reveal that the RIPK3 kinase domain exhibits catalytically independent function that is important for both RIPK3-dependent necroptosis and apoptosis.

    更新日期:2018-08-22
  • Stepwise phosphorylation of leukotriene B4 receptor 1 defines cellular responses to leukotriene B4
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-21
    Yoshimitsu Nakanishi, Modong Tan, Takako Ichiki, Asuka Inoue, Jun-ichi Yoshihara, Naoto Maekawa, Itsuki Takenoshita, Keisuke Yanagida, Shinya Yamahira, Satoshi Yamaguchi, Junken Aoki, Teruyuki Nagamune, Takehiko Yokomizo, Takao Shimizu, Motonao Nakamura

    Leukotriene B4 (LTB4) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB4. Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB4, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1. Different concentrations of LTB4 induced distinct phosphorylation events, and these ligand-induced modifications facilitated additional phosphorylation events at the basal phosphorylation sites. Because neutrophils migrate toward inflammatory sites along a gradient of LTB4, the degree of BLT1 phosphorylation likely increases in parallel with the increase in LTB4 concentration as the cells migrate. At high concentrations of LTB4, deficiencies in these two types of phosphorylation events impaired chemotaxis and β-hexosaminidase release, a proxy for degranulation, in Chinese hamster ovary (CHO-K1) and rat basophilic leukemia (RBL-2H3) cells, respectively. These results suggest that an LTB4 gradient around inflammatory sites enhances BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of phagocytic and immune cells and the initiation of local responses, including degranulation.

    更新日期:2018-08-22
  • Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-21
    Alexander I. Salter, Richard G. Ivey, Jacob J. Kennedy, Valentin Voillet, Anusha Rajan, Eva J. Alderman, Uliana J. Voytovich, Chenwei Lin, Daniel Sommermeyer, Lingfeng Liu, Jeffrey R. Whiteaker, Raphael Gottardo, Amanda G. Paulovich, Stanley R. Riddell

    Chimeric antigen receptors (CARs) link an antigen recognition domain to intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3ζ or 4-1BB/CD3ζ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function, clinical efficacy, and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8+ CD28/CD3ζ and 4-1BB/CD3ζ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3ζ CARs activated faster and larger-magnitude changes in protein phosphorylation, which correlated with an effector T cell–like phenotype and function. In contrast, 4-1BB/CD3ζ CAR T cells preferentially expressed T cell memory–associated genes and exhibited sustained antitumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3ζ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor and that signal strength is a key determinant of T cell fate. Thus, tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.

    更新日期:2018-08-22
  • G protein–coupled receptor kinases (GRKs) orchestrate biased agonism at the β2-adrenergic receptor
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-21
    Minjung Choi, Dean P. Staus, Laura M. Wingler, Seungkirl Ahn, Biswaranjan Pani, William D. Capel, Robert J. Lefkowitz

    Biased agonists of G protein–coupled receptors (GPCRs), which selectively activate either G protein– or β-arrestin–mediated signaling pathways, are of major therapeutic interest because they have the potential to show improved efficacy and specificity as drugs. Efforts to understand the mechanistic basis of this phenomenon have focused on the hypothesis that G proteins and β-arrestins preferentially couple to distinct GPCR conformations. However, because GPCR kinase (GRK)–dependent receptor phosphorylation is a critical prerequisite for the recruitment of β-arrestins to most GPCRs, GRKs themselves may play an important role in establishing biased signaling. We showed that an alanine mutant of the highly conserved residue tyrosine 219 (Y219A) in transmembrane domain five of the β2-adrenergic receptor (β2AR) was incapable of β-arrestin recruitment, receptor internalization, and β-arrestin–mediated activation of extracellular signal–regulated kinase (ERK), whereas it retained the ability to signal through G protein. We found that the impaired β-arrestin recruitment in cells was due to reduced GRK-mediated phosphorylation of the β2AR Y219A C terminus, which was recapitulated in vitro with purified components. Furthermore, in vitro ligation of a synthetically phosphorylated peptide onto the C terminus of β2AR Y219A rescued both the initial recruitment of β-arrestin and its engagement with the intracellular core of the receptor. These data suggest that the Y219A mutation generates a G protein–biased state primarily by conformational selection against GRK coupling, rather than against β-arrestin. Together, these findings highlight the importance of GRKs in modulating the biased agonism of GPCRs.

    更新日期:2018-08-22
  • Identification of Toll-like receptor signaling inhibitors based on selective activation of hierarchically acting signaling proteins
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-14
    Sirish K. Ippagunta, Julie A. Pollock, Naina Sharma, Wenwei Lin, Taosheng Chen, Kazuki Tawaratsumida, Anthony A. High, Jaeki Min, Yizhe Chen, R. Kiplin Guy, Vanessa Redecke, John A. Katzenellenbogen, Hans Häcker

    Toll-like receptors (TLRs) recognize various pathogen- and host tissue–derived molecules and initiate inflammatory immune responses. Exaggerated or prolonged TLR activation, however, can lead to etiologically diverse diseases, such as bacterial sepsis, metabolic and autoimmune diseases, or stroke. Despite the apparent medical need, no small-molecule drugs against TLR pathways are clinically available. This may be because of the complex signaling mechanisms of TLRs, which are governed by a series of protein-protein interactions initiated by Toll/interleukin-1 receptor homology domains (TIR) found in TLRs and the cytoplasmic adaptor proteins TIRAP and MyD88. Oligomerization of TLRs with MyD88 or TIRAP leads to the recruitment of members of the IRAK family of kinases and the E3 ubiquitin ligase TRAF6. We developed a phenotypic drug screening system based on the inducible homodimerization of either TIRAP, MyD88, or TRAF6, that ranked hits according to their hierarchy of action. From a bioactive compound library, we identified methyl-piperidino-pyrazole (MPP) as a TLR-specific inhibitor. Structure-activity relationship analysis, quantitative proteomics, protein-protein interaction assays, and cellular thermal shift assays suggested that MPP targets the TIR domain of MyD88. Chemical evolution of the original MPP scaffold generated compounds with selectivity for distinct TLRs that interfered with specific TIR interactions. Administration of an MPP analog to mice protected them from TLR4-dependent inflammation. These results validate this phenotypic screening approach and suggest that the MPP scaffold could serve as a starting point for the development of anti-inflammatory drugs.

    更新日期:2018-08-15
  • Inflammasome SUMOylation
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-14
    Erin R. Williams

    NLRP3 inflammasome activity is inhibited by SUMOylation.

    更新日期:2018-08-15
  • Mitotic phosphorylation regulates Hsp72 spindle localization by uncoupling ATP binding from substrate release
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-14
    Manjeet Mukherjee, Sarah Sabir, Laura O’Regan, Josephina Sampson, Mark W. Richards, Nicolas Huguenin-Dezot, James R. Ault, Jason W. Chin, Anastasia Zhuravleva, Andrew M. Fry, Richard Bayliss

    Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

    更新日期:2018-08-15
  • Aurora-PLK1 cascades as key signaling modules in the regulation of mitosis
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-14
    Vladimir Joukov, Arcangela De Nicolo

    Mitosis is controlled by reversible protein phosphorylation involving specific kinases and phosphatases. A handful of major mitotic protein kinases, such as the cyclin B–CDK1 complex, the Aurora kinases, and Polo-like kinase 1 (PLK1), cooperatively regulate distinct mitotic processes. Research has identified proteins and mechanisms that integrate these kinases into signaling cascades that guide essential mitotic events. These findings have important implications for our understanding of the mechanisms of mitotic regulation and may advance the development of novel antimitotic drugs. We review collected evidence that in vertebrates, the Aurora kinases serve as catalytic subunits of distinct complexes formed with the four scaffold proteins Bora, CEP192, INCENP, and TPX2, which we deem “core” Aurora cofactors. These complexes and the Aurora-PLK1 cascades organized by Bora, CEP192, and INCENP control crucial aspects of mitosis and all pathways of spindle assembly. We compare the mechanisms of Aurora activation in relation to the different spindle assembly pathways and draw a functional analogy between the CEP192 complex and the chromosomal passenger complex that may reflect the coevolution of centrosomes, kinetochores, and the actomyosin cleavage apparatus. We also analyze the roles and mechanisms of Aurora-PLK1 signaling in the cell and centrosome cycles and in the DNA damage response.

    更新日期:2018-08-15
  • Functional coupling of GABAA/B receptors and the channel TRPV4 mediates rapid progesterone signaling in the oviduct
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-14
    Carole Jung, Victor Fernández-Dueñas, Cristina Plata, Anna Garcia-Elias, Francisco Ciruela, José M. Fernández-Fernández, Miguel A. Valverde

    The molecular mechanism by which progesterone (P4) modulates the transport of ova and embryos along the oviduct is not fully resolved. We report a rapid response to P4 and agonists of γ-aminobutyric acid receptors A and B (GABAA/B) in the mouse oviduct that was characterized by oscillatory Ca2+ signals and increased ciliary beat frequency (CBF). Pharmacological manipulation, genetic ablation, and siRNA-mediated knockdown in oviductal cells, as well as overexpression experiments in HEK 293T cells, confirmed the participation of the cationic channel TRPV4, different subunits of GABAA (α1 to α3, β2, and β3), and GABAB1 in P4-induced responses. TRPV4-mediated Ca2+ entry in close proximity to the inositol trisphosphate receptor was required to initiate and maintain Ca2+ oscillations after P4 binding to GABAA and transactivation of Gi/o protein–coupled GABAB receptors. Coimmunoprecipitation experiments and imaging of native tissue and HEK 293T cells demonstrated the close association of GABAA and GABAB1 receptors and the activation of Gi/o proteins in response to P4 and GABA receptor agonists, confirming a molecular mechanism in which P4 and GABAergic agonists cooperatively stimulate cilial beating.

    更新日期:2018-08-15
  • G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-07
    Jo-Hao Ho, Edward L. Stahl, Cullen L. Schmid, Sarah M. Scarry, Jeffrey Aubé, Laura M. Bohn

    Biased agonists of G protein–coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the κ-opioid receptor (KOR) in a manner that favors G protein coupling over β-arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and β-arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. We further explored the influence of cellular context on biased agonism at KOR ligand–directed signaling toward G protein pathways over β-arrestin–dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein–biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [35S]GTPγS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism.

    更新日期:2018-08-10
  • The acid-sensing ion channel ASIC1a mediates striatal synapse remodeling and procedural motor learning
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-07
    Zhe Yu, Yan-Jiao Wu, Yi-Zhi Wang, Di-Shi Liu, Xing-Lei Song, Qin Jiang, Ying Li, Siyu Zhang, Nan-Jie Xu, Michael Xi Zhu, Wei-Guang Li, Tian-Le Xu

    Acid-sensing ion channel 1a (ASIC1a) is abundant in multiple brain regions, including the striatum, which serves as the input nucleus of the basal ganglia and is critically involved in procedural learning and motor memory. We investigated the functional role of ASIC1a in striatal neurons. We found that ASIC1a was critical for striatum-dependent motor coordination and procedural learning by regulating the synaptic plasticity of striatal medium spiny neurons. Global deletion of Asic1a in mice led to increased dendritic spine density but impaired spine morphology and postsynaptic architecture, which were accompanied by the decreased function of N-methyl-d-aspartate (NMDA) receptors at excitatory synapses. These structural and functional changes caused by the loss of ASIC1a were largely mediated by reduced activation (phosphorylation) of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal–regulated protein kinases (ERKs). Consequently, Asic1a null mice exhibited poor performance on multiple motor tasks, which was rescued by striatal-specific expression of either ASIC1a or CaMKII. Together, our data reveal a previously unknown mechanism mediated by ASIC1a that promotes the excitatory synaptic function underlying striatum-related procedural learning and memory.

    更新日期:2018-08-10
  • Profiling the origin, dynamics, and function of traction force in B cell activation
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-07
    Junyi Wang, Feng Lin, Zhengpeng Wan, Xiaolin Sun, Yun Lu, Jianyong Huang, Fei Wang, Yingyue Zeng, Ying-Hua Chen, Yan Shi, Wenjie Zheng, Zhanguo Li, Chunyang Xiong, Wanli Liu

    B lymphocytes use B cell receptors (BCRs) to recognize membrane-bound antigens to further initiate cell spreading and contraction responses during B cell activation. We combined traction force microscopy and live-cell imaging to profile the origin, dynamics, and function of traction force generation in these responses. We showed that B cell activation required the generation of 10 to 20 nN of traction force when encountering antigens presented by substrates with stiffness values from 0.5 to 1 kPa, which mimic the rigidity of antigen-presenting cells in vivo. Perturbation experiments revealed that F-actin remodeling and myosin- and dynein-mediated contractility contributed to traction force generation and B cell activation. Moreover, membrane-proximal BCR signaling molecules (including Lyn, Syk, Btk, PLC-γ2, BLNK, and Vav3) and adaptor molecules (Grb2, Cbl, and Dok-3) linking BCR microclusters and motor proteins were also required for the sustained generation of these traction forces. We found a positive correlation between the strength of the traction force and the mean fluorescence intensity of the BCR microclusters. Furthermore, we demonstrated that isotype-switched memory B cells expressing immunoglobulin G (IgG)–BCRs generated greater traction forces than did mature naïve B cells expressing IgM-BCRs during B cell activation. Last, we observed that primary B cells from patients with rheumatoid arthritis generated greater traction forces than did B cells from healthy donors in response to antigen stimulation. Together, these data delineate the origin, dynamics, and function of traction force during B cell activation.

    更新日期:2018-08-10
  • Starving cancer cells to death
    Sci. Signal. (IF 6.378) Pub Date : 2018-08-07
    Annalisa M. VanHook

    Cisplatin-resistant cancer cells are particularly susceptible to starvation-induced death.

    更新日期:2018-08-10
  • Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-31
    Wayne Stallaert, Yannick Brüggemann, Ola Sabet, Lisa Baak, Marina Gattiglio, Philippe I. H. Bastiaens

    The ability of cells to adapt their response to growth factors in relation to their environment is an essential aspect of tissue development and homeostasis. We found that signaling mediated by the Eph family of receptor tyrosine kinases from cell-cell contacts changed the cellular response to the growth factor EGF by modulating the vesicular trafficking of its receptor, EGFR. Eph receptor activation trapped EGFR in Rab5-positive early endosomes by inhibiting Akt-dependent vesicular recycling. By altering the spatial distribution of EGFR activity, EGF-promoted Akt signaling from the plasma membrane was suppressed, thereby inhibiting cell migration. In contrast, ERK signaling from endosomal EGFR was preserved to maintain a proliferative response to EGF stimulation. We also found that soluble extracellular signals engaging the G protein–coupled receptor Kiss1 (Kiss1R) similarly suppressed EGFR vesicular recycling to inhibit EGF-promoted migration. Eph or Kiss1R activation also suppressed EGF-promoted migration in Pten−/− mouse embryonic fibroblasts, which exhibit increased constitutive Akt activity, and in MDA-MB-231 triple-negative breast cancer cells, which overexpress EGFR. The cellular environment can thus generate context-dependent responses to EGF stimulation by modulating EGFR vesicular trafficking dynamics.

    更新日期:2018-08-01
  • Caught in the “Akt”: Cross-talk between EphA2 and EGFR through the Akt-PIKfyve axis maintains cellular sensitivity to EGF
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-31
    Xiaojun Shi, Bingcheng Wang

    Activation of EGFR (epidermal growth factor receptor) and Eph receptor often exerts opposing effects on cell functions. In this issue of Science Signaling, Stallaert et al. reveal how cells maintain sustained response to EGF stimulation by replenishing EGFR at the plasma membrane and how conflicting signals from the EphA-ephrin system and EGFR are integrated to coordinate cellular responses, including cell migration and proliferation.

    更新日期:2018-08-01
  • Cholesterol sulfate is a DOCK2 inhibitor that mediates tissue-specific immune evasion in the eye
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-31
    Tetsuya Sakurai, Takehito Uruno, Yuki Sugiura, Takaaki Tatsuguchi, Kazuhiko Yamamura, Miho Ushijima, Yuko Hattori, Mutsuko Kukimoto-Niino, Chiemi Mishima-Tsumagari, Mayuki Watanabe, Makoto Suematsu, Yoshinori Fukui

    Although immune responses are essential to protect the body from infection, they can also harm tissues. Certain tissues and organs, including the eye, constitute specialized microenvironments that locally inhibit immune reactivity. Dedicator of cytokinesis protein 2 (DOCK2) is a Rac-specific guanine nucleotide exchange factor (GEF) that is predominantly found in hematopoietic cells. DOCK2 plays a key role in immune surveillance because it is essential for the activation and migration of leukocytes. DOCK2 mutations cause severe immunodeficiency in humans. We found that DOCK2-mediated Rac activation and leukocyte migration were effectively inhibited by cholesterol sulfate (CS), but not by cholesterol or other sulfated steroids. CS bound to the catalytic domain of DOCK2 and suppressed its GEF activity. Mass spectrometric quantification revealed that CS was most abundantly produced in the Harderian gland, which provides the lipids that form the oily layer of the tear film. Sulfation of cholesterol is mediated by the sulfotransferases SULT2B1b and, to a lesser extent, SULT2B1a, which are produced from the same gene through alternative splicing. By genetically inactivating Sult2b1, we showed that the lack of CS in mice augmented ultraviolet- and antigen-induced ocular surface inflammation, which was suppressed by administration of eye drops containing CS. Thus, CS is a naturally occurring DOCK2 inhibitor and contributes to the generation of the immunosuppressive microenvironment in the eye.

    更新日期:2018-08-01
  • CDK12-mediated transcriptional regulation of noncanonical NF-κB components is essential for signaling
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-31
    Kate L. Henry, Debra Kellner, Bekim Bajrami, John E. Anderson, Mercedes Beyna, Govinda Bhisetti, Tom Cameron, Andrew G. Capacci, Andrea Bertolotti-Ciarlet, Jun Feng, Benbo Gao, Brian Hopkins, Tracy Jenkins, Kejie Li, Tricia May-Dracka, Paramasivam Murugan, Ru Wei, Weike Zeng, Norm Allaire, Alan Buckler, Christine Loh, Peter Juhasz, Brian Lucas, Katelin A. Ennis, Elisabeth Vollman, Ellen Cahir-McFarland, Erik C. Hett, Michelle L. Ols

    Members of the family of nuclear factor κB (NF-κB) transcription factors are critical for multiple cellular processes, including regulating innate and adaptive immune responses, cell proliferation, and cell survival. Canonical NF-κB complexes are retained in the cytoplasm by the inhibitory protein IκBα, whereas noncanonical NF-κB complexes are retained by p100. Although activation of canonical NF-κB signaling through the IκBα kinase complex is well studied, few regulators of the NF-κB–inducing kinase (NIK)–dependent processing of noncanonical p100 to p52 and the subsequent nuclear translocation of p52 have been identified. We discovered a role for cyclin-dependent kinase 12 (CDK12) in transcriptionally regulating the noncanonical NF-κB pathway. High-content phenotypic screening identified the compound 919278 as a specific inhibitor of the lymphotoxin β receptor (LTβR), and tumor necrosis factor (TNF) receptor superfamily member 12A (FN14)–dependent nuclear translocation of p52, but not of the TNF-α receptor–mediated nuclear translocation of p65. Chemoproteomics identified CDK12 as the target of 919278. CDK12 inhibition by 919278, the CDK inhibitor THZ1, or siRNA-mediated knockdown resulted in similar global transcriptional changes and prevented the LTβR- and FN14-dependent expression of MAP3K14 (which encodes NIK) as well as NIK accumulation by reducing phosphorylation of the carboxyl-terminal domain of RNA polymerase II. By coupling a phenotypic screen with chemoproteomics, we identified a pathway for the activation of the noncanonical NF-κB pathway that could serve as a therapeutic target in autoimmunity and cancer.

    更新日期:2018-08-01
  • Supplementing leukemia therapy
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-31
    John F. Foley

    Increasing the amount of dietary histidine enhances the sensitivity of leukemia xenografts in mice to methotrexate.

    更新日期:2018-08-01
  • Modeling chemotherapy-induced stress to identify rational combination therapies in the DNA damage response pathway
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-24
    Ozan Alkan, Birgit Schoeberl, Millie Shah, Alexander Koshkaryev, Tim Heinemann, Daryl C. Drummond, Michael B. Yaffe, Andreas Raue

    Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multiscale computational model that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated on extensive data from U2OS osteosarcoma cells, including the cell cycle distribution of the initial cell population, signaling data measured by Western blotting, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model predicted the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we confirmed experimentally. Computational models such as the one presented here can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death and to rationally identify chemotherapy-potentiating drug combinations.

    更新日期:2018-07-25
  • Mechanisms of inside-out signaling of the high-affinity IgG receptor FcγRI
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-24
    Arianne M. Brandsma, Samantha L. Schwartz, Michael J. Wester, Christopher C. Valley, Gittan L. A. Blezer, Gestur Vidarsson, Keith A. Lidke, Toine ten Broeke, Diane S. Lidke, Jeanette H. W. Leusen

    Fc receptors (FcRs) are an important bridge between the innate and adaptive immune system. Fc gamma receptor I (FcγRI; CD64), the high-affinity receptor for immunoglobulin G (IgG), plays roles in inflammation, autoimmune responses, and immunotherapy. Stimulation of myeloid cells with cytokines, such as tumor necrosis factor–α ( TNFα) and interferon-γ ( IFNγ), increases the binding of FcγRI to immune complexes (ICs), such as antibody-opsonized pathogens or tumor cells, through a process known as “inside-out” signaling. Using super-resolution imaging, we found that stimulation of cells with IL-3 also enhanced the clustering of FcγRI both before and after exposure to ICs. This increased clustering was dependent on an intact actin cytoskeleton. We found that chemical inhibition of the activity of the phosphatase PP1 reduced FcγRI inside-out signaling, although the phosphorylation of FcγRI itself was unaffected. Furthermore, the antibody-dependent cytotoxic activity of human neutrophils toward CD20-expressing tumor cells was increased after stimulation with TNFα and IFNγ. These results suggest that nanoscale reorganization of FcγRI, stimulated by cytokine-induced, inside-out signaling, enhances FcγRI cellular effector functions.

    更新日期:2018-07-25
  • The transcription factor Lef1 switches partners from β-catenin to Smad3 during muscle stem cell quiescence
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-24
    Ajoy Aloysius, Ramanuj DasGupta, Jyotsna Dhawan

    Skeletal muscle stem cells (MuSCs), also known as satellite cells, persist in adult mammals by entering a state of quiescence (G0) during the early postnatal period. Quiescence is reversed during damage-induced regeneration and re-established after regeneration. Entry of cultured myoblasts into G0 is associated with a specific, reversible induction of Wnt target genes, thus implicating members of the Tcf and Lef1 (Tcf/Lef) transcription factor family, which mediate transcriptional responses to Wnt signaling, in the initiation of quiescence. We found that the canonical Wnt effector β-catenin, which cooperates with Tcf/Lef, was dispensable for myoblasts to enter quiescence. Using pharmacological and genetic approaches in cultured C2C12 myoblasts and in MuSCs, we demonstrated that Tcf/Lef activity during quiescence depended not on β-catenin but on the transforming growth factor–β (TGF-β) effector and transcriptional coactivator Smad3, which colocalized with Lef1 at canonical Wnt-responsive elements and directly interacted with Lef1 specifically in G0. Depletion of Smad3, but not β-catenin, reduced Lef1 occupancy at target promoters, Tcf/Lef target gene expression, and self-renewal of myoblasts. In vivo, MuSCs underwent a switch from β-catenin–Lef1 to Smad3-Lef1 interactions during the postnatal switch from proliferation to quiescence, with β-catenin–Lef1 interactions recurring during damage-induced reactivation. Our findings suggest that the interplay of Wnt-Tcf/Lef and TGF-β–Smad3 signaling activates canonical Wnt target promoters in a manner that depends on β-catenin during myoblast proliferation but is independent of β-catenin during MuSC quiescence.

    更新日期:2018-07-25
  • Quantitative analysis of competitive cytokine signaling predicts tissue thresholds for the propagation of macrophage activation
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-24
    James Bagnall, Christopher Boddington, Hazel England, Ruth Brignall, Polly Downton, Zainab Alsoufi, James Boyd, William Rowe, Alexander Bennett, Catherine Walker, Antony Adamson, Nisha M. X. Patel, Ronan O’Cualain, Lorraine Schmidt, David G. Spiller, Dean A. Jackson, Werner Müller, Mark Muldoon, Michael R. H. White, Pawel Paszek

    Toll-like receptor (TLR) signaling regulates macrophage activation and effector cytokine propagation in the constrained environment of a tissue. In macrophage populations, TLR4 stimulates the dose-dependent transcription of nuclear factor κB (NF-κB) target genes. However, using single-RNA counting, we found that individual cells exhibited a wide range (three orders of magnitude) of expression of the gene encoding the proinflammatory cytokine tumor necrosis factor–α (TNF-α). The TLR4-induced TNFA transcriptional response correlated with the extent of NF-κB signaling in the cells and their size. We compared the rates of TNF-α production and uptake in macrophages and mouse embryonic fibroblasts and generated a mathematical model to explore the heterogeneity in the response of macrophages to TLR4 stimulation and the propagation of the TNF-α signal in the tissue. The model predicts that the local propagation of the TLR4-dependent TNF-α response and cellular NF-κB signaling are limited to small distances of a few cell diameters between neighboring tissue-resident macrophages. In our predictive model, TNF-α propagation was constrained by competitive uptake of TNF-α from the environment, rather than by heterogeneous production of the cytokine. We propose that the highly constrained architecture of tissues enables effective localized propagation of inflammatory cues while avoiding out-of-context responses at longer distances.

    更新日期:2018-07-25
  • Lysosomes on an acid trip
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-24
    Wei Wong

    Acidic environments cause lysosomes to redistribute to the cell periphery, suppressing mTORC1, translation, and circadian oscillations.

    更新日期:2018-07-25
  • Unique BIR domain sets determine inhibitor of apoptosis protein–driven cell death and NOD2 complex signal specificity
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-17
    Steven M. Chirieleison, Joseph K. Rathkey, Derek W. Abbott

    The mammalian IAPs, X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2), play pivotal roles in innate immune signaling and inflammatory homeostasis, often working in parallel or in conjunction at a signaling complex. IAPs direct both nucleotide-binding oligomerization domain-containing 2 (NOD2) signaling complexes and cell death mechanisms to appropriately regulate inflammation. Although it is known that XIAP is critical for NOD2 signaling and that the loss of cIAP1 and cIAP2 blunts NOD2 activity, it is unclear whether these three highly related proteins can compensate for one another in NOD2 signaling or in mechanisms governing apoptosis or necroptosis. This potential redundancy is critically important, given that genetic loss of XIAP causes both very early onset inflammatory bowel disease and X-linked lymphoproliferative syndrome 2 (XLP-2) and that the overexpression of cIAP1 and cIAP2 is linked to both carcinogenesis and chemotherapeutic resistance. Given the therapeutic interest in IAP inhibition and the potential toxicities associated with disruption of inflammatory homeostasis, we used synthetic biology techniques to examine the functional redundancies of key domains in the IAPs. From this analysis, we defined the features of the IAPs that enable them to function at overlapping signaling complexes but remain independent and functionally exclusive in their roles as E3 ubiquitin ligases in innate immune and inflammatory signaling.

    更新日期:2018-07-18
  • Synthetic lethality of TNK2 inhibition in PTPN11-mutant leukemia
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-17
    Chelsea Jenkins, Samuel B. Luty, Julia E. Maxson, Christopher A. Eide, Melissa L. Abel, Corinne Togiai, Eneida R. Nemecek, Daniel Bottomly, Shannon K. McWeeney, Beth Wilmot, Marc Loriaux, Bill H. Chang, Jeffrey W. Tyner

    The protein tyrosine phosphatase PTPN11 is implicated in the pathogenesis of juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and other malignancies. Activating mutations in PTPN11 increase downstream proliferative signaling and cell survival. We investigated the signaling upstream of PTPN11 in JMML and AML cells and found that PTPN11 was activated by the nonreceptor tyrosine/serine/threonine kinase TNK2 and that PTPN11-mutant JMML and AML cells were sensitive to TNK2 inhibition. In cultured human cell–based assays, PTPN11 and TNK2 interacted directly, enabling TNK2 to phosphorylate PTPN11, which subsequently dephosphorylated TNK2 in a negative feedback loop. Mutations in PTPN11 did not affect this physical interaction but increased the basal activity of PTPN11 such that TNK2-mediated activation was additive. Consequently, coexpression of TNK2 and mutant PTPN11 synergistically increased mitogen-activated protein kinase (MAPK) signaling and enhanced colony formation in bone marrow cells from mice. Chemical inhibition of TNK2 blocked MAPK signaling and colony formation in vitro and decreased disease burden in a patient with PTPN11-mutant JMML who was treated with the multikinase (including TNK2) inhibitor dasatinib. Together, these data suggest that TNK2 is a promising therapeutic target for PTPN11-mutant leukemias.

    更新日期:2018-07-18
  • CRISPR, cancer, and p53
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-17
    Leslie K. Ferrarelli

    CRISPR-Cas9 genome editing is most efficient in cells lacking functional p53 protein, a phenotype common to cancer cells.

    更新日期:2018-07-18
  • Multisite phosphorylation is required for sustained interaction with GRKs and arrestins during rapid μ-opioid receptor desensitization
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-17
    Elke Miess, Arisbel B. Gondin, Arsalan Yousuf, Ralph Steinborn, Nadja Mösslein, Yunshi Yang, Martin Göldner, Julia G. Ruland, Moritz Bünemann, Cornelius Krasel, MacDonald J. Christie, Michelle L. Halls, Stefan Schulz, Meritxell Canals

    G protein receptor kinases (GRKs) and β-arrestins are key regulators of μ-opioid receptor (MOR) signaling and trafficking. We have previously shown that high-efficacy opioids such as DAMGO stimulate a GRK2/3-mediated multisite phosphorylation of conserved C-terminal tail serine and threonine residues, which facilitates internalization of the receptor. In contrast, morphine-induced phosphorylation of MOR is limited to Ser375 and is not sufficient to drive substantial receptor internalization. We report how specific multisite phosphorylation controlled the dynamics of GRK and β-arrestin interactions with MOR and show how such phosphorylation mediated receptor desensitization. We showed that GRK2/3 was recruited more quickly than was β-arrestin to a DAMGO-activated MOR. β-Arrestin recruitment required GRK2 activity and MOR phosphorylation, but GRK recruitment also depended on the phosphorylation sites in the C-terminal tail, specifically four serine and threonine residues within the 370TREHPSTANT379 motif. Our results also suggested that other residues outside this motif participated in the initial and transient recruitment of GRK and β-arrestins. We identified two components of high-efficacy agonist desensitization of MOR: a sustained component, which required GRK2-mediated phosphorylation and a potential soluble factor, and a rapid component, which was likely mediated by GRK2 but independent of receptor phosphorylation. Elucidating these complex receptor-effector interactions represents an important step toward a mechanistic understanding of MOR desensitization that leads to the development of tolerance and dependence.

    更新日期:2018-07-18
  • Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-10
    Yichong Zhang, Ji-Hoon Lee, Tanya T. Paull, Sarah Gehrke, Angelo D’Alessandro, Qianhui Dou, Vadim N. Gladyshev, Elizabeth A. Schroeder, Samantha K. Steyl, Brooke E. Christian, Gerald S. Shadel

    Mitochondria are integral to cellular energy metabolism and ATP production and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS), which not only can damage cellular components but also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease ataxia-telangiectasia (A-T), is a key player in the nuclear DNA damage response. However, ATM also performs a redox-sensing function mediated through formation of ROS-dependent disulfide-linked dimers. We found that mitochondria-derived hydrogen peroxide promoted ATM dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with the separation of its redox and DNA damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox sensing–deficient C2991L mutant revealed that one function of ATM redox sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the abundance and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. We propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.

    更新日期:2018-07-12
  • New connections: Putting the brakes on inflammation
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-10
    Erin R. Williams

    By targeting multiple pathways, microRNAs limit inflammation during sepsis.

    更新日期:2018-07-12
  • The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+ T cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-10
    Guillaume Gaud, Romain Roncagalli, Karima Chaoui, Isabelle Bernard, Julien Familiades, Céline Colacios, Sahar Kassem, Bernard Monsarrat, Odile Burlet-Schiltz, Anne Gonzalez de Peredo, Bernard Malissen, Abdelhadi Saoudi

    The activation of T cells requires the guanine nucleotide exchange factor VAV1. Using mice in which a tag for affinity purification was attached to endogenous VAV1 molecules, we analyzed by quantitative mass spectrometry the signaling complex that assembles around activated VAV1. Fifty VAV1-binding partners were identified, most of which had not been previously reported to participate in VAV1 signaling. Among these was CD226, a costimulatory molecule of immune cells. Engagement of CD226 induced the tyrosine phosphorylation of VAV1 and synergized with T cell receptor (TCR) signals to specifically enhance the production of interleukin-17 (IL-17) by primary human CD4+ T cells. Moreover, co-engagement of the TCR and a risk variant of CD226 that is associated with autoimmunity (rs763361) further enhanced VAV1 activation and IL-17 production. Thus, our study reveals that a VAV1-based, synergistic cross-talk exists between the TCR and CD226 during both physiological and pathological T cell responses and provides a rational basis for targeting CD226 for the management of autoimmune diseases.

    更新日期:2018-07-12
  • IKK promotes cytokine-induced and cancer-associated AMPK activity and attenuates phenformin-induced cell death in LKB1-deficient cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-10
    Ricardo J. Antonia, Albert S. Baldwin

    The 5′ AMP-activated protein kinase (AMPK) is an energy sensor that is activated upon phosphorylation of Thr172 in its activation loop by the kinase LKB1, CAMKK2, or TAK1. TAK1-dependent AMPK phosphorylation of Thr172 is less well characterized than phosphorylation of this site by LKB1 or CAMKK2. An important target of TAK1 is IκB kinase (IKK), which controls the activation of the transcription factor NF-κB. We tested the hypothesis that IKK acted downstream of TAK1 to activate AMPK by phosphorylating Thr172. IKK was required for the phosphorylation of Thr172 in AMPK in response to treatment with the inflammatory cytokine IL-1β or TNF-α or upon TAK1 overexpression. In addition, IKK regulated basal AMPK Thr172 phosphorylation in several cancer cell types independently of TAK1, indicating that other modes of IKK activation could stimulate AMPK. We found that IKK directly phosphorylated AMPK at Thr172 independently of the tumor suppressor LKB1 or energy stress. Accordingly, in LKB1-deficient cells, IKK inhibition reduced AMPK Thr172 phosphorylation in response to the mitochondrial inhibitor phenformin. This response led to enhanced apoptosis and suggests that IKK inhibition in combination with phenformin could be used clinically to treat patients with LKB1-deficient cancers.

    更新日期:2018-07-12
  • Evolution of human, chicken, alligator, frog, and zebrafish mineralocorticoid receptors: Allosteric influence on steroid specificity
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-03
    Yoshinao Katsu, Kaori Oka, Michael E. Baker

    Although multiple steroid ligands of the glucocorticoid, mineralocorticoid, and progestin families bind to and regulate the activity of mineralocorticoid receptors (MRs), the responses to these ligands differ across species. To understand how the different domains of MRs contribute to the ligand-induced activation or inhibition of MR activity, we studied the response to eight steroids (aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, corticosterone, progesterone, 19-norprogesterone, and spironolactone) of human, chicken, alligator, frog, and zebrafish full-length MRs and truncated MRs, which lacked the N-terminal domain (NTD) and DNA binding domain (DBD). Compared to full-length MRs, some truncated MRs were not activated by the steroids, and others required higher steroid concentrations for activation. Progesterone, 19-norprogesterone, and spironolactone did not activate full-length or truncated human, alligator, or frog MRs. However, at 10 nM, these steroids activated full-length chicken and zebrafish MRs, whereas at 100 nM, these steroids had little activity for truncated chicken MRs, but they retained activity for truncated zebrafish MRs. This suggests that regulation of the activation of the chicken MR by progestin resides in the NTD-DBD and that of the zebrafish MR resides in the hinge-LBD. Zebrafish and chicken MRs contain a serine corresponding to Ser810 in human MR, which is required for the antagonist activity of progesterone for human MR, suggesting a previously uncharacterized mechanism of regulation of progestin activation of chicken and zebrafish MRs. These findings suggest that progesterone may be a physiological activator of chicken and zebrafish MRs.

    更新日期:2018-07-04
  • Proteomic analysis of S-nitrosylated nuclear proteins in rat cortical neurons
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-03
    Jacob G. Smith, Sarah G. Aldous, Catia Andreassi, Giovanni Cuda, Marco Gaspari, Antonella Riccio

    Neurons modulate gene expression in response to extrinsic signals to enable brain development, cognition, and learning and to process stimuli that regulate systemic physiological functions. This signal-to-gene communication is facilitated by posttranslational modifications such as S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, but few other nuclear targets of S-nitrosylation have been identified to date. We used S-nitrosothiol resin-assisted capture on NO donor-treated nuclear extracts from rat cortical neurons and identified 614 S-nitrosylated nuclear proteins. Of these, 131 proteins have not previously been shown to be S-nitrosylated in any system, and 555 are previously unidentified targets of S-nitrosylation in neurons. The sites of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines were found at 33% of these sites. In addition, lysine motifs were necessary for promoting the S-nitrosylation of HDAC2 and methyl-CpG binding protein 3 (MBD3). Moreover, S-nitrosylation of the histone-binding protein RBBP7 was necessary for dendritogenesis of cortical neurons in culture. Together, our findings characterize S-nitrosylated nuclear proteins in neurons and identify S-nitrosylation motifs that may be shared with other targets of NO signaling.

    更新日期:2018-07-04
  • Tomosyn functions as a PKCδ-regulated fusion clamp in mast cell degranulation
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-03
    Iris K. Madera-Salcedo, Luca Danelli, Neeraj Tiwari, Bárbara Dema, Emeline Pacreau, Shamila Vibhushan, Joëlle Birnbaum, Chantal Agabriel, Valérie Liabeuf, Caroline Klingebiel, Gaël Menasche, Marina Macias-Silva, Marc Benhamou, Nicolas Charles, Claudia González-Espinosa, Joana Vitte, Ulrich Blank

    Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) family proteins mediate membrane fusion critical for vesicular transport and cellular secretion. Mast cells rely on SNARE-mediated membrane fusion for degranulation stimulated by crosslinking of immunoglobulin E (IgE) bound to the Fcε receptor (FcεRI). We investigated the mechanisms downstream of receptor activation that control degranulation. We found that the SNARE binding protein tomosyn-1 (also known as STXBP5) inhibited FcεRI-stimulated degranulation of mast cells. After mast cell activation, tomosyn-1 was phosphorylated on serine and threonine residues, dissociated from the SNARE protein syntaxin 4 (STX4), and associated with STX3. We identified PKCδ as the major kinase required for tomosyn-1 threonine phosphorylation and for regulation of the interaction with STXs. Incubation with high IgE concentrations increased tomosyn-1 abundance in cultured mast cells. Similarly, in basophils from allergic patients with high amounts of serum IgE, the abundance of tomosyn-1 was increased as compared to that in patients with normal IgE concentrations. Our findings identified tomosyn-1 as an inhibitor of mast cell degranulation that required PKCδ to switch its interaction with STX partners during fusion. We suggest that the IgE-mediated increase in tomosyn-1 abundance in allergic patients may represent a counterregulatory mechanism to limit disease development.

    更新日期:2018-07-04
  • Inhibition of the acetyltransferase NAT10 normalizes progeric and aging cells by rebalancing the Transportin-1 nuclear import pathway
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-03
    Delphine Larrieu, Emmanuelle Viré, Samuel Robson, Sophia Y. Breusegem, Tony Kouzarides, Stephen P. Jackson

    Hutchinson-Gilford progeria syndrome (HGPS) is an incurable premature aging disease. Identifying deregulated biological processes in HGPS might thus help define novel therapeutic strategies. Fibroblasts from HGPS patients display defects in nucleocytoplasmic shuttling of the GTP-bound form of the small GTPase Ran (RanGTP), which leads to abnormal transport of proteins into the nucleus. We report that microtubule stabilization in HGPS cells sequestered the nonclassical nuclear import protein Transportin-1 (TNPO1) in the cytoplasm, thus affecting the nuclear localization of its cargo, including the nuclear pore protein NUP153. Consequently, nuclear Ran, nuclear anchorage of the nucleoporin TPR, and chromatin organization were disrupted, deregulating gene expression and inducing senescence. Inhibiting N-acetyltransferase 10 (NAT10) ameliorated HGPS phenotypes by rebalancing the nuclear to cytoplasmic ratio of TNPO1. This restored nuclear pore complex integrity and nuclear Ran localization, thereby correcting HGPS cellular phenotypes. We observed a similar mechanism in cells from healthy aged individuals. This study identifies a nuclear import pathway affected in aging and underscores the potential for NAT10 inhibition as a possible therapeutic strategy for HGPS and perhaps also for pathologies associated with normal aging.

    更新日期:2018-07-04
  • Sunshine for your mind
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-03
    Annalisa M. VanHook

    Moderate exposure to ultraviolet light stimulates glutamate synthesis in the brain and improves learning and memory.

    更新日期:2018-07-04
  • Nuclear import pathway key to rescuing dominant progerin phenotypes
    Sci. Signal. (IF 6.378) Pub Date : 2018-07-03
    Katherine L. Wilson

    In this issue of Science Signaling, Larrieu et al. show that an acetyltransferase inhibitor that rescues many dominant nuclear phenotypes caused by progerin, a truncated form of lamin A, does so by releasing the specialized nuclear import receptor TNPO1 from sequestration by microtubules. This release enables TNPO1-dependent import of specific cargoes, including the nuclear pore protein Nup153 and the heterogeneous nuclear ribonucleoprotein hnRNPA1, thus restoring the functionality of nuclear pore complexes, rebalancing the nucleocytoplasmic Ran gradient, and normalizing gene expression.

    更新日期:2018-07-04
  • MEG3-4 is a miRNA decoy that regulates IL-1β abundance to initiate and then limit inflammation to prevent sepsis during lung infection
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-26
    Rongpeng Li, Lizhu Fang, Qinqin Pu, Huimin Bu, Pengcheng Zhu, Zihan Chen, Min Yu, Xuefeng Li, Timothy Weiland, Arvind Bansal, Shui Qing Ye, Yuquan Wei, Jianxin Jiang, Min Wu

    Long noncoding RNAs (lncRNAs) regulate gene expression. We investigated the role of lncRNAs in the inflammatory response to bacterial infection in the lungs. We identified the lncRNA MEG3 as a tissue-specific modulator of inflammatory responses during bacterial infection. Among the 10 transcript isoforms of MEG3, transcript 4 (referred to as MEG3-4) encodes the isoform with the lowest abundance in mouse lungs. Nonetheless, we found that MEG3-4 bound to the microRNA miR-138 in a competitive manner with mRNA encoding the proinflammatory cytokine interleukin-1β (IL-1β), thereby increasing IL-1β abundance and intensifying inflammatory responses to bacterial infection in alveolar macrophages and lung epithelial cells in culture and in lung tissue in mice. MEG3-4–mediated sponging of miR-138 in the cytoplasm increased the autocrine activity of IL-1β that subsequently induced a negative feedback mechanism mediated by nuclear factor κB that decreased MEG3-4 abundance and inflammatory cytokine production. This timely reduction in MEG3-4 abundance tempered proinflammatory responses in mice with pulmonary bacterial infection, preventing the progression to sepsis. Together, these findings reveal that MEG3-4 dynamically modulates pulmonary inflammatory responses through transcriptional regulation of immune response genes, extending the decoy and sponge mechanism associated with lncRNAs to antibacterial immunity, which affects both response and disease progression.

    更新日期:2018-06-27
  • The plant cell wall integrity maintenance and immune signaling systems cooperate to control stress responses in Arabidopsis thaliana
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-26
    Timo Engelsdorf, Nora Gigli-Bisceglia, Manikandan Veerabagu, Joseph F. McKenna, Lauri Vaahtera, Frauke Augstein, Dieuwertje Van der Does, Cyril Zipfel, Thorsten Hamann

    Cell walls surround all plant cells, and their composition and structure are modified in a tightly controlled, adaptive manner to meet sometimes opposing functional requirements during growth and development. The plant cell wall integrity (CWI) maintenance mechanism controls these functional modifications, as well as responses to cell wall damage (CWD). We investigated how the CWI system mediates responses to CWD in Arabidopsis thaliana. CWD induced by cell wall–degrading enzymes or an inhibitor of cellulose biosynthesis elicited similar, turgor-sensitive stress responses. Phenotypic clustering with 27 genotypes identified a core group of receptor-like kinases (RLKs) and ion channels required for the activation of CWD responses. A genetic analysis showed that the RLK FEI2 and the plasma membrane–localized mechanosensitive Ca2+ channel MCA1 functioned downstream of the RLK THE1 in CWD perception. In contrast, pattern-triggered immunity (PTI) signaling components, including the receptors for plant elicitor peptides (AtPeps) PEPR1 and PEPR2, repressed responses to CWD. CWD induced the expression of PROPEP1 and PROPEP3, which encode the precursors of AtPep1 and AtPep3, and the release of PROPEP3 into the growth medium. Application of AtPep1 and AtPep3 repressed CWD-induced phytohormone accumulation in a concentration-dependent manner. These results suggest that AtPep-mediated signaling suppresses CWD-induced defense responses controlled by the CWI mechanism. This suppression was alleviated when PTI signaling downstream of PEPR1 and PEPR2 was impaired. Defense responses controlled by the CWI maintenance mechanism might thus compensate to some extent for the loss of PTI signaling elements.

    更新日期:2018-06-27
  • New connections: Reprogramming B cell metabolism
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-26
    John F. Foley

    Two key regulators of B cell metabolism are required for effective immune responses.

    更新日期:2018-06-27
  • Thyroid hormone receptor and ERRα coordinately regulate mitochondrial fission, mitophagy, biogenesis, and function
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-26
    Brijesh K. Singh, Rohit A. Sinha, Madhulika Tripathi, Arturo Mendoza, Kenji Ohba, Jann A. C. Sy, Sherwin Y. Xie, Jin Zhou, Jia Pei Ho, Ching-yi Chang, Yajun Wu, Vincent Giguère, Boon-Huat Bay, Jean-Marc Vanacker, Sujoy Ghosh, Karine Gauthier, Anthony N. Hollenberg, Donald P. McDonnell, Paul M. Yen

    Thyroid hormone receptor β1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and β-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA, ULK1, DRP1, or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.

    更新日期:2018-06-27
  • Inhibition of somatosensory mechanotransduction by annexin A6
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-19
    Ramin Raouf, Stéphane Lolignier, Jane E. Sexton, Queensta Millet, Sonia Santana-Varela, Anna Biller, Alice M. Fuller, Vanessa Pereira, Jyoti S. Choudhary, Mark O. Collins, Stephen E. Moss, Richard Lewis, Julie Tordo, Els Henckaerts, Michael Linden, John N. Wood

    Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron–derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein. Annexin A6–deficient mice showed increased sensitivity to mechanical stimuli. Sensory neurons from these mice showed increased activity of the cation channel Piezo2, which mediates a rapidly adapting mechano-gated current linked to proprioception and touch, and a decrease in mechanically activated, slowly adapting currents. Conversely, overexpression of annexin A6 in sensory neurons inhibited rapidly adapting currents that were partially mediated by Piezo2. Furthermore, overexpression of annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis, a disease in which mechanically evoked pain is particularly problematic. These data suggest that annexin A6 can be exploited to inhibit chronic mechanical pain.

    更新日期:2018-06-20
  • Oligomerization of MrgC11 and μ-opioid receptors in sensory neurons enhances morphine analgesia
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-19
    Shao-Qiu He, Qian Xu, Vinod Tiwari, Fei Yang, Michael Anderson, Zhiyong Chen, Shaness A. Grenald, Srinivasa N. Raja, Xinzhong Dong, Yun Guan

    The μ-opioid receptor (MOR) agonist morphine is commonly used for pain management, but it has severe adverse effects and produces analgesic tolerance. Thus, alternative ways of stimulating MOR activity are needed. We found that MrgC11, a sensory neuron–specific G protein–coupled receptor, may form heteromeric complexes with MOR. Peptide-mediated activation of MrgC11 enhanced MOR recycling by inducing coendocytosis and sorting of MOR for membrane reinsertion. MrgC11 activation also inhibited the coupling of MOR to β-arrestin-2 and enhanced the morphine-dependent inhibition of cAMP production. Intrathecal coadministration of a low dose of an MrgC agonist potentiated acute morphine analgesia and reduced chronic morphine tolerance in wild-type mice but not in Mrg-cluster knockout (Mrg KO) mice. BAM22, a bivalent agonist of MrgC and opioid receptors, enhanced the interaction between MrgC11 and MOR and produced stronger analgesia than did the individual monovalent agonists. Morphine-induced neuronal and pain inhibition was reduced in Mrg KO mice compared to that in wild-type mice. Our results uncover MrgC11-MOR interactions that lead to positive functional modulation of MOR. MrgC shares genetic homogeneity and functional similarity with human MrgX1. Thus, harnessing this positive modulation of MOR function by Mrg signaling may enhance morphine analgesia in a sensory neuron–specific fashion to limit central side effects.

    更新日期:2018-06-20
  • New connections: Engineering DNA repair vulnerability to treat cancer
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-19
    Leslie K. Ferrarelli

    Drugs that impair DNA repair broaden cancer sensitivity to common therapies.

    更新日期:2018-06-20
  • PARP12 suppresses Zika virus infection through PARP-dependent degradation of NS1 and NS3 viral proteins
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-19
    Lili Li, Hui Zhao, Ping Liu, Chunfeng Li, Natalie Quanquin, Xue Ji, Nina Sun, Peishuang Du, Cheng-Feng Qin, Ning Lu, Genhong Cheng

    Zika virus infection stimulates a type I interferon (IFN) response in host cells, which suppresses viral replication. Type I IFNs exert antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). To screen for antiviral ISGs that restricted Zika virus replication, we individually knocked out 21 ISGs in A549 lung cancer cells and identified PARP12 as a strong inhibitor of Zika virus replication. Our findings suggest that PARP12 mediated the ADP-ribosylation of NS1 and NS3, nonstructural viral proteins that are involved in viral replication and modulating host defense responses. This modification of NS1 and NS3 triggered their proteasome-mediated degradation. These data increase our understanding of the antiviral activity of PARP12 and suggest a molecular basis for the potential development of therapeutics against Zika virus.

    更新日期:2018-06-20
  • SOCE mediated by STIM and Orai is essential for pacemaker activity in the interstitial cells of Cajal in the gastrointestinal tract
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-12
    Haifeng Zheng, Bernard T. Drumm, Scott Earley, Tae Sik Sung, Sang Don Koh, Kenton M. Sanders

    Electrical pacemaker activity generates phasic contractions and motility patterns such as segmentation and peristalsis in the gastrointestinal tract. Pacemaker currents are generated in interstitial cells of Cajal (ICC), which release Ca2+ from intracellular stores that stimulates Ca2+-activated Cl− channels (CaCCs) in the plasma membrane. Thus, Ca2+ stores must be maintained to sustain pacemaker activity. Store-operated Ca2+ entry (SOCE) facilitates the refilling of Ca2+ stores by a mechanism dependent upon interactions between STIM and Orai proteins. We investigated the role of SOCE in ICC pacemaker activity. Reintroduction of extracellular Ca2+ in store-depleted ICC resulted in CaCC activation. Blocking CaCCs revealed an inwardly rectifying current with properties of a Ca2+ release–activated current (ICRAC). An inhibitory peptide that interfered with the STIM-Orai interaction blocked ICRAC in HEK 293 cells expressing STIM1 and Orai1 and blocked spontaneous transient inward currents (STICs) and slow wave currents in ICC. STICs, which are fundamental pacemaker events in ICC, were blocked by an Orai antagonist. Imaging of Ca2+ transients linked to pacemaker activity in ICC in intact muscles showed that the Orai antagonist blocked Ca2+ transients in ICC. These data suggest that Ca2+ recovery through STIM-Orai interactions is necessary to maintain ICC pacemaker activity.

    更新日期:2018-06-13
  • “Disruptor” residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Gα subunits
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-12
    Ali Asli, Isra Sadiya, Meirav Avital-Shacham, Mickey Kosloff

    Understanding the molecular basis of interaction specificity between RGS (regulator of G protein signaling) proteins and heterotrimeric (αβγ) G proteins would enable the manipulation of RGS-G protein interactions, explore their functions, and effectively target them therapeutically. RGS proteins are classified into four subfamilies (R4, R7, RZ, and R12) and function as negative regulators of G protein signaling by inactivating Gα subunits. We found that the R12 subfamily members RGS10 and RGS14 had lower activity than most R4 subfamily members toward the Gi subfamily member Gαo. Using structure-based energy calculations with multiple Gα-RGS complexes, we identified R12-specific residues in positions that are predicted to determine the divergent activity of this subfamily. This analysis predicted that these residues, which we call “disruptor residues,” interact with the Gα helical domain. We engineered the R12 disruptor residues into the RGS domains of the high-activity R4 subfamily and found that these altered proteins exhibited reduced activity toward Gαo. Reciprocally, replacing the putative disruptor residues in RGS18 (a member of the R4 subfamily that exhibited low activity toward Gαo) with the corresponding residues from a high-activity R4 subfamily RGS protein increased its activity toward Gαo. Furthermore, the high activity of the R4 subfamily toward Gαo was independent of the residues in the homologous positions to the R12 subfamily and RGS18 disruptor residues. Thus, our results suggest that the identified RGS disruptor residues function as negative design elements that attenuate RGS activity for specific Gα proteins.

    更新日期:2018-06-13
  • A painful takedown of host defenses
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-12
    Wei Wong

    A flesh-eating bacterium disables host defenses by inducing pain-sensing neurons to release a neutrophil-inhibiting peptide.

    更新日期:2018-06-13
  • A coupled-clock system drives the automaticity of human sinoatrial nodal pacemaker cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-12
    Kenta Tsutsui, Oliver J. Monfredi, Syevda G. Sirenko-Tagirova, Larissa A. Maltseva, Rostislav Bychkov, Mary S. Kim, Bruce D. Ziman, Kirill V. Tarasov, Yelena S. Tarasova, Jing Zhang, Mingyi Wang, Alexander V. Maltsev, Jaclyn A. Brennan, Igor R. Efimov, Michael D. Stern, Victor A. Maltsev, Edward G. Lakatta

    The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca2+ releases generated by a Ca2+ clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca2+–cAMP–protein kinase A (PKA) signaling regulated clock coupling. When these clocks became uncoupled, SANCs failed to generate spontaneous action potentials, showing a depolarized membrane potential and disorganized local Ca2+ releases that failed to activate the M clock. β-Adrenergic receptor (β-AR) stimulation, which increases cAMP concentrations and clock coupling in other species, restored spontaneous, rhythmic action potentials in some nonbeating “arrested” human SANCs by increasing intracellular Ca2+ concentrations and synchronizing diastolic local Ca2+ releases. When β-AR stimulation was withdrawn, the clocks again became uncoupled, and SANCs reverted to a nonbeating arrested state. Thus, automaticity of human pacemaker cells is driven by a coupled-clock system driven by Ca2+-cAMP-PKA signaling. Extreme clock uncoupling led to failure of spontaneous action potential generation, which was restored by recoupling of the clocks. Clock coupling and action potential firing in some of these arrested cells can be restored by β-AR stimulation–induced augmentation of Ca2+-cAMP-PKA signaling.

    更新日期:2018-06-13
  • Regulation of thymocyte trafficking by Tagap, a GAP domain protein linked to human autoimmunity
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-12
    Jonathan S. Duke-Cohan, Yuki Ishikawa, Akihiro Yoshizawa, Young-Il Choi, Chin-Nien Lee, Oreste Acuto, Stephan Kissler, Ellis L. Reinherz

    Multiple autoimmune pathologies are associated with single-nucleotide polymorphisms of the human gene TAGAP, which encodes TAGAP, a guanosine triphosphatase (GTPase)–activating protein. We showed in mice that Tagap-mediated signaling by the sema3E/plexin-D1 ligand-receptor complex attenuates thymocytes’ adhesion to the cortex through their β1-containing integrins. By promoting thymocyte detachment within the cortex of the thymus, Tagap-mediated signaling enabled their translocation to the medulla, which is required for continued thymic selection. Tagap physically interacted with the cytoplasmic domain of plexin-D1 and directly stimulated the activity and signaling of the GTPase RhoA. In addition, Tagap indirectly mediated the activation of Cdc42 in response to the binding of sema3E to plexin-D1. Both RhoA and Cdc42 are key mediators of cytoskeletal and integrin dynamics in thymocytes. Knockdown of Tagap in mice suppressed the sema3E- and plexin-D1–mediated release of thymocytes that adhered within the cortex through β1-containing integrins. This suppression led to the impaired translocation of thymocytes from the cortex to the medulla and resulted in the formation of ectopic medullary structures within the thymic cortex. Our results suggest that TAGAP variation modulates the risk of autoimmunity by altering thymocyte migration during thymic selection.

    更新日期:2018-06-13
  • Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-05
    Marta Portela, Liu Yang, Sayantanee Paul, Xia Li, Alexey Veraksa, Linda M. Parsons, Helena E. Richardson

    Epithelial cell polarity is linked to the control of tissue growth and tumorigenesis. The tumor suppressor and cell polarity protein lethal-2-giant larvae (Lgl) promotes Hippo signaling and inhibits Notch signaling to restrict tissue growth in Drosophila melanogaster. Notch signaling is greater in lgl mutant tissue than in wild-type tissue because of increased acidification of endosomal vesicles, which promotes the proteolytic processing and activation of Notch by γ-secretase. We showed that the increased Notch signaling and tissue growth defects of lgl mutant tissue depended on endosomal vesicle acidification mediated by the vacuolar adenosine triphosphatase (V-ATPase). Lgl promoted the activity of the V-ATPase by interacting with Vap33 (VAMP-associated protein of 33 kDa). Vap33 physically and genetically interacted with Lgl and V-ATPase subunits and repressed V-ATPase–mediated endosomal vesicle acidification and Notch signaling. Vap33 overexpression reduced the abundance of the V-ATPase component Vha44, whereas Lgl knockdown reduced the binding of Vap33 to the V-ATPase component Vha68-3. Our data indicate that Lgl promotes the binding of Vap33 to the V-ATPase, thus inhibiting V-ATPase–mediated endosomal vesicle acidification and thereby reducing γ-secretase activity, Notch signaling, and tissue growth. Our findings implicate the deregulation of Vap33 and V-ATPase activity in polarity-impaired epithelial cancers.

    更新日期:2018-06-06
  • The ion channel TRPM7 is required for B cell lymphopoiesis
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-05
    Mithunah Krishnamoorthy, Fathima Hifza Mohamed Buhari, Tiantian Zhao, Patrick M. Brauer, Kyle Burrows, Eric Yixiao Cao, Vincent Moxley-Paquette, Arthur Mortha, Juan Carlos Zúñiga-Pflücker, Bebhinn Treanor

    The transient receptor potential (TRP) family is a large family of widely expressed ion channels that regulate the intracellular concentration of ions and metals and respond to various chemical and physical stimuli. TRP subfamily M member 7 (TRPM7) is unusual in that it contains both an ion channel and a kinase domain. TRPM7 is a divalent cation channel with preference for Ca2+ and Mg2+. It is required for the survival of DT40 cells, a B cell line; however, deletion of TRPM7 in T cells does not impair their development. We found that expression of TRPM7 was required for B cell development in mice. Mice that lacked TRPM7 in B cells failed to generate peripheral B cells because of a developmental block at the pro-B cell stage. The loss of TRPM7 kinase activity alone did not affect the proportion of peripheral mature B cells or the development of B cells in the bone marrow. However, supplementation with a high concentration of extracellular Mg2+ partially rescued the development of TRPM7-deficient B cells in vitro. Thus, our findings identify a critical role for TRPM7 ion channel activity in B cell development.

    更新日期:2018-06-06
  • Highlight: Distinct functions of the ion channel–kinase TRPM7
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-05
    Erin Williams

    Two studies in this week’s issue of Science Signaling identify distinct activities of the ion channel–kinase TRPM7 that control discrete functions in B cells.

    更新日期:2018-06-06
  • The channel-kinase TRPM7 regulates antigen gathering and internalization in B cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-05
    Mithunah Krishnamoorthy, Laabiah Wasim, Fathima Hifza Mohamed Buhari, Tiantian Zhao, Trisha Mahtani, Josephine Ho, Sohee Kang, Francina Deason-Towne, Anne-Laure Perraud, Carsten Schmitz, Bebhinn Treanor

    Members of the transient receptor potential (TRP) family of ion channels are cellular sensors involved in numerous physiological and pathological processes. We identified the TRP subfamily M member 7 (TRPM7) channel-kinase as a previously uncharacterized regulator of B cell activation. We showed that TRPM7 played a critical role in the early events of B cell activation through both its ion channel and kinase functions. DT40 B cells deficient in TRPM7 or expressing a kinase-deficient mutant of TRPM7 showed defective gathering of antigen and prolonged B cell receptor (BCR) signaling. We showed that lipid metabolism was altered in TRPM7-deficient cells and in cells expressing a kinase-deficient mutant of TRPM7 and suggest that PLC-γ2 may be a target of the kinase activity of TRPM7. Primary B cells that expressed less TRPM7 or were treated with a pharmacological inhibitor of TRPM7 also displayed defective antigen gathering and increased BCR signaling. Finally, we demonstrated that blocking TRPM7 function compromised antigen internalization and presentation to T cells. These data suggest that TRPM7 controls an essential process required for B cell affinity maturation and the production of high-affinity antibodies.

    更新日期:2018-06-06
  • K63-linked polyubiquitin chains bind to DNA to facilitate DNA damage repair
    Sci. Signal. (IF 6.378) Pub Date : 2018-06-05
    Pengda Liu, Wenjian Gan, Siyuan Su, Arthur V. Hauenstein, Tian-min Fu, Bradley Brasher, Carsten Schwerdtfeger, Anthony C. Liang, Ming Xu, Wenyi Wei

    Polyubiquitylation is canonically viewed as a posttranslational modification that governs protein stability or protein-protein interactions, in which distinct polyubiquitin linkages ultimately determine the fate of modified protein(s). We explored whether polyubiquitin chains have any nonprotein-related function. Using in vitro pull-down assays with synthetic materials, we found that polyubiquitin chains with the Lys63 (K63) linkage bound to DNA through a motif we called the “DNA-interacting patch” (DIP), which is composed of the adjacent residues Thr9, Lys11, and Glu34. Upon DNA damage, the binding of K63-linked polyubiquitin chains to DNA enhanced the recruitment of repair factors through their interaction with an Ile44 patch in ubiquitin to facilitate DNA repair. Furthermore, experimental or cancer patient–derived mutations within the DIP impaired the DNA binding capacity of ubiquitin and subsequently attenuated K63-linked polyubiquitin chain accumulation at sites of DNA damage, thereby resulting in defective DNA repair and increased cellular sensitivity to DNA-damaging agents. Our results therefore highlight a critical physiological role for K63-linked polyubiquitin chains in binding to DNA to facilitate DNA damage repair.

    更新日期:2018-06-06
  • R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes
    Sci. Signal. (IF 6.378) Pub Date : 2018-05-29
    Pilar Mendoza, Nuria Martínez-Martín, Elena R. Bovolenta, Diana Reyes-Garau, Pablo Hernansanz-Agustín, Pilar Delgado, Manuel D. Diaz-Muñoz, Clara L. Oeste, Isabel Fernández-Pisonero, Ester Castellano, Antonio Martínez-Ruiz, Diego Alonso-Lopez, Eugenio Santos, Xosé R. Bustelo, Tomohiro Kurosaki, Balbino Alarcón

    Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell–intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies.

    更新日期:2018-05-30
  • Thrombospondin-1 promotes matrix homeostasis by interacting with collagen and lysyl oxidase precursors and collagen cross-linking sites
    Sci. Signal. (IF 6.378) Pub Date : 2018-05-29
    Silvia Rosini, Nicholas Pugh, Arkadiusz M. Bonna, David J. S. Hulmes, Richard W. Farndale, Josephine C. Adams

    Fibrillar collagens of the extracellular matrix are critical for tissue structure and physiology; however, excessive or abnormal deposition of collagens is a defining feature of fibrosis. Regulatory mechanisms that act on collagen fibril assembly potentially offer new targets for antifibrotic treatments. Tissue weakening, altered collagen fibril morphologies, or both, are shared phenotypes of mice lacking matricellular thrombospondins. Thrombospondin-1 (TSP1) plays an indirect role in collagen homeostasis through interactions with matrix metalloproteinases and transforming growth factor–β1 (TGF-β1). We found that TSP1 also affects collagen fibril formation directly. Compared to skin from wild-type mice, skin from Thbs1−/− mice had reduced collagen cross-linking and reduced prolysyl oxidase (proLOX) abundance with increased conversion to catalytically active LOX. In vitro, TSP1 bound to both the C-propeptide domain of collagen I and the highly conserved KGHR sequences of the collagen triple-helical domain that participate in cross-linking. TSP1 also bound to proLOX and inhibited proLOX processing by bone morphogenetic protein-1. In human dermal fibroblasts (HDFs), TSP1 and collagen I colocalized in intracellular vesicles and on extracellular collagen fibrils, whereas TSP1 and proLOX colocalized only in intracellular vesicles. Inhibition of LOX-mediated collagen cross-linking did not prevent the extracellular association between collagen and TSP1; however, treatment of HDFs with KGHR-containing, TSP1-binding, triple-helical peptides disrupted the collagen-TSP1 association, perturbed the collagen extracellular matrix, and increased myofibroblastic differentiation in a manner that depended on TGF-β receptor 1. Thus, the extracellular KGHR-dependent interaction of TSP1 with fibrillar collagens contributes to fibroblast homeostasis.

    更新日期:2018-05-30
  • Bacterial manipulation of host cell metabolism
    Sci. Signal. (IF 6.378) Pub Date : 2018-05-29
    Annalisa M. VanHook

    A Helicobacter pylori exotoxin manipulates host cell metabolism by inducing amino acid starvation and inhibiting mTORC1 signaling.

    更新日期:2018-05-30
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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