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  • EGFR signaling moves infection
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-12
    Erin R. Williams

    A viral EGF homolog increases the migration of cells infected with vaccinia virus to promote viral spread.

    更新日期:2019-02-13
  • Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-12
    Marc Herb, Alexander Gluschko, Katja Wiegmann, Alina Farid, Anne Wolf, Olaf Utermöhlen, Oleg Krut, Martin Krönke, Michael Schramm

    A major function of macrophages during infection is initiation of the proinflammatory response, leading to the secretion of cytokines that help to orchestrate the immune response. Here, we identify reactive oxygen species (ROS) as crucial mediators of proinflammatory signaling leading to cytokine secretion in Listeria monocytogenes–infected macrophages. ROS produced by NADPH oxidases (Noxes), such as Nox2, are key components of the macrophage response to invading pathogens; however, our data show that the ROS that mediated proinflammatory signaling were produced by mitochondria (mtROS). We identified the inhibitor of κB (IκB) kinase (IKK) complex regulatory subunit NEMO [nuclear factor κB (NF-κB) essential modulator] as a target for mtROS. Specifically, mtROS induced intermolecular covalent linkage of NEMO through disulfide bonds formed by Cys54 and Cys347, which was essential for activation of the IKK complex and subsequent signaling through the extracellular signal–regulated protein kinases 1 and 2 (ERK1/2) and NF-κB pathways that eventually led to the secretion of proinflammatory cytokines. We thus identify mtROS-dependent disulfide linkage of NEMO as an essential regulatory step of the proinflammatory response of macrophages to bacterial infection.

    更新日期:2019-02-13
  • Inflammation, necrosis, and the kinase RIP3 are key mediators of AAG-dependent alkylation-induced retinal degeneration
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-12
    Mariacarmela Allocca, Joshua J. Corrigan, Aprotim Mazumder, Kimberly R. Fake, Leona D. Samson

    DNA-alkylating agents are commonly used to kill cancer cells, but the base excision repair (BER) pathway they trigger can also produce toxic intermediates that cause tissue damage, such as retinal degeneration (RD). Apoptosis, a process of programmed cell death, is assumed to be the main mechanism of this alkylation-induced photoreceptor (PR) cell death in RD. Here, we studied the involvement of necroptosis (another programmed cell death process) and inflammation in alkylation-induced RD. Male mice exposed to a methylating agent exhibited a reduced number of PR cell rows, active gliosis, and cytokine induction and macrophage infiltration in the retina. Dying PRs exhibited a necrotic morphology, increased 8-hydroxyguanosine abundance (an oxidative damage marker), and overexpression of the necroptosis-associated genes Rip1 and Rip3. The activity of PARP1, which mediates BER, cell death, and inflammation, was increased in PR cells and associated with the release of proinflammatory chemokine HMGB1 from PR nuclei. Mice lacking the anti-inflammatory cytokine IL-10 exhibited more severe RD, whereas deficiency of RIP3 (also known as RIPK3) conferred partial protection. Female mice were partially protected from alkylation-induced RD, showing reduced necroptosis and inflammation compared to males. PRs in mice lacking the BER-initiating DNA glycosylase AAG did not exhibit alkylation-induced necroptosis or inflammation. Our findings show that AAG-initiated BER at alkylated DNA bases induces sex-dependent RD primarily by triggering necroptosis and activating an inflammatory response that amplifies the original damage and, furthermore, reveal new potential targets to prevent this side effect of chemotherapy.

    更新日期:2019-02-13
  • An immunoproteomic approach to characterize the CAR interactome and signalosome
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-12
    Maria C. Ramello, Ismahène Benzaïd, Brent M. Kuenzi, Maritza Lienlaf-Moreno, Wendy M. Kandell, Daniel N. Santiago, Mibel Pabón-Saldaña, Lancia Darville, Bin Fang, Uwe Rix, Sean Yoder, Anders Berglund, John M. Koomen, Eric B. Haura, Daniel Abate-Daga

    Adoptive transfer of T cells that express a chimeric antigen receptor (CAR) is an approved immunotherapy that may be curative for some hematological cancers. To better understand the therapeutic mechanism of action, we systematically analyzed CAR signaling in human primary T cells by mass spectrometry. When we compared the interactomes and the signaling pathways activated by distinct CAR-T cells that shared the same antigen-binding domain but differed in their intracellular domains and their in vivo antitumor efficacy, we found that only second-generation CARs induced the expression of a constitutively phosphorylated form of CD3ζ that resembled the endogenous species. This phenomenon was independent of the choice of costimulatory domains, or the hinge/transmembrane region. Rather, it was dependent on the size of the intracellular domains. Moreover, the second-generation design was also associated with stronger phosphorylation of downstream secondary messengers, as evidenced by global phosphoproteome analysis. These results suggest that second-generation CARs can activate additional sources of CD3ζ signaling, and this may contribute to more intense signaling and superior antitumor efficacy that they display compared to third-generation CARs. Moreover, our results provide a deeper understanding of how CARs interact physically and/or functionally with endogenous T cell molecules, which will inform the development of novel optimized immune receptors.

    更新日期:2019-02-13
  • Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-05
    Arnaud Augert, Emily Eastwood, Ali H. Ibrahim, Nan Wu, Eli Grunblatt, Ryan Basom, Denny Liggitt, Keith D. Eaton, Renato Martins, John T. Poirier, Charles M. Rudin, Francesca Milletti, Wei-Yi Cheng, Fiona Mack, David MacPherson

    Small cell lung cancer (SCLC) is a recalcitrant, aggressive neuroendocrine-type cancer for which little change to first-line standard-of-care treatment has occurred within the last few decades. Unlike nonsmall cell lung cancer (NSCLC), SCLC harbors few actionable mutations for therapeutic intervention. Lysine-specific histone demethylase 1A (LSD1 also known as KDM1A) inhibitors were previously shown to have selective activity in SCLC models, but the underlying mechanism was elusive. Here, we found that exposure to the selective LSD1 inhibitor ORY-1001 activated the NOTCH pathway, resulting in the suppression of the transcription factor ASCL1 and the repression of SCLC tumorigenesis. Our analyses revealed that LSD1 bound to the NOTCH1 locus, thereby suppressing NOTCH1 expression and downstream signaling. Reactivation of NOTCH signaling with the LSD1 inhibitor reduced the expression of ASCL1 and neuroendocrine cell lineage genes. Knockdown studies confirmed the pharmacological inhibitor-based results. In vivo, sensitivity to LSD1 inhibition in SCLC patient-derived xenograft (PDX) models correlated with the extent of consequential NOTCH pathway activation and repression of a neuroendocrine phenotype. Complete and durable tumor regression occurred with ORY-1001–induced NOTCH activation in a chemoresistant PDX model. Our findings reveal how LSD1 inhibitors function in this tumor and support their potential as a new and targeted therapy for SCLC.

    更新日期:2019-02-06
  • Interrogating B cell signaling pathways: A quest for novel therapies for mantle cell lymphoma
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-05
    Makhdum Ahmed, Elizabeth Lorence, Jeffrey Wang, Dayoung Jung, Liang Zhang, Krystle Nomie, Michael Wang

    Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma that is largely chemoresistant. Ibrutinib, a drug that inhibits Bruton’s tyrosine kinase (BTK), has improved the overall survival of patients with MCL; however, resistance to ibrutinib has emerged as a decisive, negative factor in the prognosis of MCL. Adopting a more patient-centric therapeutic approach that incorporates applied genomics and interrogation of B cell signaling pathways may offer an alternative route to reach durable remission in patients with MCL. Although targeting genetic variants in MCL is not yet feasible in the clinical setting, the identification and targeting of increasingly active B cell signaling pathways may be a viable therapeutic strategy that may improve patient outcomes. Genome-editing tools and sequencing platforms could play dominant roles in patient-centric approaches of treatment in the future, potentially improving clinical outcomes for patients with MCL.

    更新日期:2019-02-06
  • Inhibition of T cell activation and function by the adaptor protein CIN85
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-05
    Mei Suen Kong, Akiko Hashimoto-Tane, Yusuke Kawashima, Machie Sakuma, Tadashi Yokosuka, Kohei Kometani, Reiko Onishi, Nick Carpino, Osamu Ohara, Tomohiro Kurosaki, Kia Kien Phua, Takashi Saito

    T cell activation is initiated by signaling molecules downstream of the T cell receptor (TCR) that are organized by adaptor proteins. CIN85 (Cbl-interacting protein of 85 kDa) is one such adaptor protein. Here, we showed that CIN85 limited T cell responses to TCR stimulation. Compared to activated wild-type (WT) T cells, those that lacked CIN85 produced more IL-2 and exhibited greater proliferation. After stimulation of WT T cells with their cognate antigen, CIN85 was recruited to the TCR signaling complex. Early TCR signaling events, such as phosphorylation of ζ-chain–associated protein kinase 70 (Zap70), Src homology 2 (SH2) domain–containing leukocyte protein of 76 kDa (SLP76), and extracellular signal–regulated kinase (Erk), were enhanced in CIN85-deficient T cells. The inhibitory function of CIN85 required the SH3 and PR regions of the adaptor, which associated with the phosphatase suppressor of TCR signaling–2 (Sts-2) after TCR stimulation. Together, our data suggest that CIN85 is recruited to the TCR signaling complex and mediates inhibition of T cell activation through its association with Sts-2.

    更新日期:2019-02-06
  • Autophagy-independent p62 in metastatic melanoma
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-05
    Leslie K. Ferrarelli

    By pairing up with RNA-binding proteins, p62 promotes melanoma progression.

    更新日期:2019-02-06
  • Hypoxic cancer–associated fibroblasts increase NCBP2-AS2/HIAR to promote endothelial sprouting through enhanced VEGF signaling
    Sci. Signal. (IF 6.378) Pub Date : 2019-02-05
    Fernanda G. Kugeratski, Samuel J. Atkinson, Lisa J. Neilson, Sergio Lilla, John R. P. Knight, Jens Serneels, Amelie Juin, Shehab Ismail, David M. Bryant, Elke K. Markert, Laura M. Machesky, Massimiliano Mazzone, Owen J. Sansom, Sara Zanivan

    Intratumoral hypoxia causes the formation of dysfunctional blood vessels, which contribute to tumor metastasis and reduce the efficacy of therapeutic treatments. Blood vessels are embedded in the tumor stroma of which cancer-associated fibroblasts (CAFs) constitute a prominent cellular component. We found that hypoxic human mammary CAFs promoted angiogenesis in CAF-endothelial cell cocultures in vitro. Mass spectrometry–based proteomic analysis of the CAF secretome unraveled that hypoxic CAFs contributed to blood vessel abnormalities by altering their secretion of various pro- and anti-angiogenic factors. Hypoxia induced pronounced remodeling of the CAF proteome, including proteins that have not been previously related to this process. Among those, the uncharacterized protein NCBP2-AS2 that we renamed HIAR (hypoxia-induced angiogenesis regulator) was the protein most increased in abundance in hypoxic CAFs. Silencing of HIAR abrogated the pro-angiogenic and pro-migratory function of hypoxic CAFs by decreasing secretion of the pro-angiogenic factor VEGFA and consequently reducing VEGF/VEGFR downstream signaling in the endothelial cells. Our study has identified a regulator of angiogenesis and provides a map of hypoxia-induced molecular alterations in mammary CAFs.

    更新日期:2019-02-06
  • The pseudokinase domains of guanylyl cyclase–A and –B allosterically increase the affinity of their catalytic domains for substrate
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-29
    Aaron B. Edmund, Timothy F. Walseth, Nicholas M. Levinson, Lincoln R. Potter

    Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity. Molecular modeling indicated that all the residues of the ATP-binding site of the prototypical kinase PKA, except the catalytic aspartate, are conserved in the PKDs of GC-A and GC-B. Kinase-inactivating alanine substitutions for the invariant lysine in subdomain II or the aspartate in the DYG-loop of GC-A and GC-B failed to decrease enzyme phosphate content, consistent with the PKDs lacking kinase activity. In contrast, both mutations reduced enzyme activation by blocking the ability of ATP to decrease the Michaelis constant without affecting peptide-dependent activation. The analogous lysine-to-alanine substitution in a glutamate-substituted phosphomimetic mutant form of GC-B also reduced enzyme activity, consistent with ATP stimulating guanylyl cyclase activity through an allosteric, phosphorylation-independent mechanism. Mutations designed to rigidify the conserved regulatory or catalytic spines within the PKDs increased guanylyl cyclase activity, increased sensitivity to natriuretic peptide, or reduced the Michaelis constant in the absence of ATP, consistent with ATP binding stabilizing the PKD in a conformation analogous to that of catalytically active kinases. We conclude that allosteric mechanisms evolutionarily conserved in the PKDs promote the catalytic activation of transmembrane guanylyl cyclases.

    更新日期:2019-01-30
  • Immune suppression with complex III
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-29
    John F. Foley

    Loss of mitochondrial complex III in regulatory T cells reduces their suppressive activity without affecting their survival.

    更新日期:2019-01-30
  • The transcription factor SP3 drives TNF-α expression in response to Smac mimetics
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-29
    Shawn T. Beug, Herman H. Cheung, Tarun Sanda, Martine St-Jean, Caroline E. Beauregard, Hapsatou Mamady, Stephen D. Baird, Eric C. LaCasse, Robert G. Korneluk

    The controlled production and downstream signaling of the inflammatory cytokine tumor necrosis factor–α (TNF-α) are important for immunity and its anticancer effects. Although chronic stimulation with TNF-α is detrimental to the health of the host in several autoimmune and inflammatory disorders, TNF-α—contrary to what its name implies—leads to cancer formation by promoting cell proliferation and survival. Smac mimetic compounds (SMCs), small-molecule antagonists of inhibitor of apoptosis proteins (IAPs), switch the TNF-α signal from promoting survival to promoting death in cancer cells. Using a genome-wide siRNA screen to identify factors required for SMC–to–TNF-α–mediated cancer cell death, we identified the transcription factor SP3 as a critical molecule in both basal and SMC-induced production of TNF-α by engaging the nuclear factor κB (NF-κB) transcriptional pathway. Moreover, the promotion of TNF-α expression by SP3 activity confers differential sensitivity of cancer versus normal cells to SMC treatment. The key role of SP3 in TNF-α production and signaling will help us further understand TNF-α biology and provide insight into mechanisms relevant to cancer and inflammatory disease.

    更新日期:2019-01-30
  • Systemic analysis of tyrosine kinase signaling reveals a common adaptive response program in a HER2-positive breast cancer
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-22
    Martin Schwill, Rastislav Tamaskovic, Aaron S. Gajadhar, Florian Kast, Forest M. White, Andreas Plückthun

    Drug-induced compensatory signaling and subsequent rewiring of the signaling pathways that support cell proliferation and survival promote the development of acquired drug resistance in tumors. Here, we sought to analyze the adaptive kinase response in cancer cells after distinct treatment with agents targeting human epidermal growth factor receptor 2 (HER2), specifically those that induce either only temporary cell cycle arrest or, alternatively, apoptosis in HER2-overexpressing cancers. We compared trastuzumab, ARRY380, the combination thereof, and a biparatopic, HER2-targeted designed ankyrin repeat protein (DARPin; specifically, 6L1G) and quantified the phosphoproteome by isobaric tagging using tandem mass tag liquid chromatography/tandem mass spectrometry (TMT LC-MS/MS). We found a specific signature of persistently phosphorylated tyrosine peptides after the nonapoptotic treatments, which we used to distinguish between different treatment-induced cancer cell fates. Next, we analyzed the activation of serine/threonine and tyrosine kinases after treatment using a bait peptide chip array and predicted the corresponding active kinases. Through a combined system-wide analysis, we identified a common adaptive kinase response program that involved the activation of focal adhesion kinase 1 (FAK1), protein kinase C-δ (PRKCD), and Ephrin (EPH) family receptors. These findings reveal potential targets to prevent adaptive resistance to HER2-targeted therapies.

    更新日期:2019-01-23
  • Why geneticists stole cancer research even though cancer is primarily a signaling disease
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-22
    Michael B. Yaffe

    Genetic approaches to cancer research have dramatically advanced our understanding of the pathophysiology of this disease, leading to similar genetics-based approaches for precision therapy, which have been less successful. Reconfiguring and adapting the types of technologies that underlie genetic research to dissect tumor cell signaling in clinical samples may offer an alternative road forward.

    更新日期:2019-01-23
  • Putting the squeeze on ERK signaling
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-22
    Annalisa M. VanHook

    Cell compaction induces epithelial cell elimination by reducing ERK activation.

    更新日期:2019-01-23
  • Illuminating the dark phosphoproteome
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-22
    Elise J. Needham, Benjamin L. Parker, Timur Burykin, David E. James, Sean J. Humphrey

    Protein phosphorylation is a major regulator of protein function and biological outcomes. This was first recognized through functional biochemical experiments, and in the past decade, major technological advances in mass spectrometry have enabled the study of protein phosphorylation on a global scale. This rapidly growing field of phosphoproteomics has revealed that more than 100,000 distinct phosphorylation events occur in human cells, which likely affect the function of every protein. Phosphoproteomics has improved the understanding of the function of even the most well-characterized protein kinases by revealing new downstream substrates and biology. However, current biochemical and bioinformatic approaches have only identified kinases for less than 5% of the phosphoproteome, and functional assignments of phosphosites are almost negligible. Notably, our understanding of the relationship between kinases and their substrates follows a power law distribution, with almost 90% of phosphorylation sites currently assigned to the top 20% of kinases. In addition, more than 150 kinases do not have a single known substrate. Despite a small group of kinases dominating biomedical research, the number of substrates assigned to a kinase does not correlate with disease relevance as determined by pathogenic human mutation prevalence and mouse model phenotypes. Improving our understanding of the substrates targeted by all kinases and functionally annotating the phosphoproteome will be broadly beneficial. Advances in phosphoproteomics technologies, combined with functional screening approaches, should make it feasible to illuminate the connectivity and functionality of the entire phosphoproteome, providing enormous opportunities for discovering new biology, therapeutic targets, and possibly diagnostics.

    更新日期:2019-01-23
  • Transcriptional repressor REST drives lineage stage–specific chromatin compaction at Ptch1 and increases AKT activation in a mouse model of medulloblastoma
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-22
    Tara H. W. Dobson, Rong-Hua Tao, Jyothishmathi Swaminathan, Shinji Maegawa, Shavali Shaik, Javiera Bravo-Alegria, Ajay Sharma, Bridget Kennis, Yanwen Yang, Keri Callegari, Amanda R. Haltom, Pete Taylor, Mari Kogiso, Lin Qi, Soumen Khatua, Stewart Goldman, Rishi R. Lulla, Jason Fangusaro, Tobey J. MacDonald, Xiao-Nan Li, Cynthia Hawkins, Veena Rajaram, Vidya Gopalakrishnan

    In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-β (infants) subgroups. Neuronal maturation is greater in SHH-β than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG) wherein conditional NeuroD2-controlled REST transgene expression in lineage-committed Ptch1+/− cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context–specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1. Expression of Arrb1, which encodes β-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed RESTTG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1. These cells also had decreased expression of Pten, which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-β than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.

    更新日期:2019-01-23
  • Cadherin-11–mediated adhesion of macrophages to myofibroblasts establishes a profibrotic niche of active TGF-β
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-15
    Monika Lodyga, Elizabeth Cambridge, Henna M. Karvonen, Pardis Pakshir, Brian Wu, Stellar Boo, Melanie Kiebalo, Riitta Kaarteenaho, Michael Glogauer, Mohit Kapoor, Kjetil Ask, Boris Hinz

    Macrophages contribute to the activation of fibroblastic cells into myofibroblasts, which secrete collagen and contract the collagen matrix to acutely repair injured tissue. Persistent myofibroblast activation leads to the accumulation of fibrotic scar tissue that impairs organ function. We investigated the key processes that turn acute beneficial repair into destructive progressive fibrosis. We showed that homotypic cadherin-11 interactions promoted the specific binding of macrophages to and persistent activation of profibrotic myofibroblasts. Cadherin-11 was highly abundant at contacts between macrophages and myofibroblasts in mouse and human fibrotic lung tissues. In attachment assays, cadherin-11 junctions mediated specific recognition and strong adhesion between macrophages and myofibroblasts. One functional outcome of cadherin-11–mediated adhesion was locally restricted activation of latent transforming growth factor–β (TGF-β) between macrophage-myofibroblast pairs that was not observed in cocultures of macrophages and myofibroblasts that were not in contact with one another. Our data suggest that cadherin-11 junctions maintain latent TGF-β–producing macrophages and TGF-β–activating myofibroblasts in close proximity to one another. Inhibition of homotypic cadherin-11 interactions could be used to cause macrophage-myofibroblast separation, thereby destabilizing the profibrotic niche.

    更新日期:2019-01-16
  • The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide–dependent neutrophil recruitment
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-15
    Mariko Takahashi, Kumi Izawa, Makoto Urai, Yoshinori Yamanishi, Akie Maehara, Masamichi Isobe, Toshihiro Matsukawa, Ayako Kaitani, Ayako Takamori, Shino Uchida, Hiromichi Yamada, Masakazu Nagamine, Tomoaki Ando, Toshiaki Shimizu, Hideoki Ogawa, Ko Okumura, Yuki Kinjo, Toshio Kitamura, Jiro Kitaura

    Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b−/− mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b+ inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.

    更新日期:2019-01-16
  • mTORC1 goes sensorless
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-15
    Wei Wong

    A metabolite of leucine regulates mTORC1 through acetylation of Raptor in certain cell types and tissues.

    更新日期:2019-01-16
  • Mapping the stochastic sequence of individual ligand-receptor binding events to cellular activation: T cells act on the rare events
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-15
    Jenny J. Y. Lin, Shalini T. Low-Nam, Katherine N. Alfieri, Darren B. McAffee, Nicole C. Fay, Jay T. Groves

    T cell receptor (TCR) binding to agonist peptide major histocompatibility complex (pMHC) triggers signaling events that initiate T cell responses. This system is remarkably sensitive, requiring only a few binding events to successfully activate a cellular response. On average, activating pMHC ligands exhibit mean dwell times of at least a few seconds when bound to the TCR. However, a T cell accumulates pMHC-TCR interactions as a stochastic series of discrete, single-molecule binding events whose individual dwell times are broadly distributed. With activation occurring in response to only a handful of such binding events, individual cells are unlikely to experience the average binding time. Here, we mapped the ensemble of pMHC-TCR binding events in space and time while simultaneously monitoring cellular activation. Our findings revealed that T cell activation hinges on rare, long–dwell time binding events that are an order of magnitude longer than the average agonist pMHC-TCR dwell time. Furthermore, we observed that short pMHC-TCR binding events that were spatially correlated and temporally sequential led to cellular activation. These observations indicate that T cell antigen discrimination likely occurs by sensing the tail end of the pMHC-TCR binding dwell time distribution rather than its average properties.

    更新日期:2019-01-16
  • Phosphorylation of the phosphatase PTPROt at Tyr399 is a molecular switch that controls osteoclast activity and bone mass in vivo
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-08
    Lee Roth, Jean Wakim, Elad Wasserman, Moran Shalev, Esther Arman, Merle Stein, Vlad Brumfeld, Cari A. Sagum, Mark T. Bedford, Jan Tuckermann, Ari Elson

    Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr527 and its activating residue Tyr416. Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr399, in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr399 caused PTPROt to dephosphorylate Src at the activating site pTyr416. In contrast, phosphorylation of PTPROt at Tyr399 enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr527, thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr399 is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.

    更新日期:2019-01-09
  • LIPID MAPS: Serving the next generation of lipid researchers with tools, resources, data, and training
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-08
    Valerie B. O’Donnell, Edward A. Dennis, Michael J. O. Wakelam, Shankar Subramaniam

    Lipids are increasingly recognized as dynamic, critical metabolites affecting human physiology and pathophysiology. LIPID MAPS is a free resource dedicated to serving the lipid research community.

    更新日期:2019-01-09
  • Combination therapy is a game of strategy
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-08
    Leslie K. Ferrarelli

    For combination therapy, knowing the “how” and “when” is just as important as knowing the “what.”

    更新日期:2019-01-09
  • Manganese activates NLRP3 inflammasome signaling and propagates exosomal release of ASC in microglial cells
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-08
    Souvarish Sarkar, Dharmin Rokad, Emir Malovic, Jie Luo, Dilshan S. Harischandra, Huajun Jin, Vellareddy Anantharam, Xuemei Huang, Mechelle Lewis, Arthi Kanthasamy, Anumantha G. Kanthasamy

    Chronic, sustained inflammation underlies many pathological conditions, including neurodegenerative diseases. Divalent manganese (Mn2+) exposure can stimulate neurotoxicity by increasing inflammation. In this study, we examined whether Mn2+ activates the multiprotein NLRP3 inflammasome complex to promote neuroinflammation. Exposing activated mouse microglial cells to Mn2+ substantially augmented NLRP3 abundance, caspase-1 cleavage, and maturation of the inflammatory cytokine interleukin-1β (IL-1β). Exposure of mice to Mn2+ had similar effects in brain microglial cells. Furthermore, Mn2+ impaired mitochondrial ATP generation, basal respiratory rate, and spare capacity in microglial cells. These data suggest that Mn-induced mitochondrial defects drove the inflammasome signal amplification. We found that Mn induced cell-to-cell transfer of the inflammasome adaptor protein ASC in exosomes. Furthermore, primed microglial cells exposed to exosomes from Mn-treated mice released more IL-1β than did cells exposed to exosomes from control-treated animals. We also observed that welders exposed to manganese-containing fumes had plasma exosomes that contained more ASC than did those from a matched control group. Together, these results suggest that the divalent metal manganese acts as a key amplifier of NLRP3 inflammasome signaling and exosomal ASC release.

    更新日期:2019-01-09
  • Activation of atypical protein kinase C by sphingosine 1-phosphate revealed by an aPKC-specific activity reporter
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-01
    Taketoshi Kajimoto, Alisha D. Caliman, Irene S. Tobias, Taro Okada, Caila A. Pilo, An-Angela N. Van, J. Andrew McCammon, Shun-ichi Nakamura, Alexandra C. Newton

    Atypical protein kinase C (aPKC) isozymes are unique in the PKC superfamily in that they are not regulated by the lipid second messenger diacylglycerol, which has led to speculation about whether a different second messenger acutely controls their function. Here, using a genetically encoded reporter that we designed, aPKC-specific C kinase activity reporter (aCKAR), we found that the lipid mediator sphingosine 1-phosphate (S1P) promoted the cellular activity of aPKC. Intracellular S1P directly bound to the purified kinase domain of aPKC and relieved autoinhibitory constraints, thereby activating the kinase. In silico studies identified potential binding sites on the kinase domain, one of which was validated biochemically. In HeLa cells, S1P-dependent activation of aPKC suppressed apoptosis. Together, our findings identify a previously undescribed molecular mechanism of aPKC regulation, a molecular target for S1P in cell survival regulation, and a tool to further explore the biochemical and biological functions of aPKC.

    更新日期:2019-01-02
  • Mechanisms of postsynaptic localization of AMPA-type glutamate receptors and their regulation during long-term potentiation
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-01
    Olivia R. Buonarati, Erik A. Hammes, Jake F. Watson, Ingo H. Greger, Johannes W. Hell

    l-Glutamate is the main excitatory neurotransmitter in the brain, with postsynaptic responses to its release predominantly mediated by AMPA-type glutamate receptors (AMPARs). A critical component of synaptic plasticity involves changes in the number of responding postsynaptic receptors, which are dynamically recruited to and anchored at postsynaptic sites. Emerging findings continue to shed new light on molecular mechanisms that mediate AMPAR postsynaptic trafficking and localization. Accordingly, unconventional secretory trafficking of AMPARs occurs in dendrites, from the endoplasmic reticulum (ER) through the ER-Golgi intermediary compartment directly to recycling endosomes, independent of the Golgi apparatus. Upon exocytosis, AMPARs diffuse in the plasma membrane to reach the postsynaptic site, where they are trapped to contribute to transmission. This trapping occurs through a combination of both intracellular interactions, such as TARP (transmembrane AMPAR regulatory protein) binding to α-actinin–stabilized PSD-95, and extracellular interactions through the receptor amino-terminal domain. These anchoring mechanisms may facilitate precise receptor positioning with respect to glutamate release sites to enable efficient synaptic transmission.

    更新日期:2019-01-02
  • A posttranslational modification code for CFTR maturation is altered in cystic fibrosis
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-01
    Sandra Pankow, Casimir Bamberger, John R. Yates

    The multistep process regulating the maturation of membrane proteins in the endoplasmic reticulum (ER) and the secretory pathway is disrupted in many protein misfolding disorders. Mutations in the ion channel CFTR that impair its folding and subsequent localization to the plasma membrane cause cystic fibrosis (CF), an inherited and eventually lethal disease that impairs the function of multiple organs, mostly the lungs. Here, we found that proper maturation of CFTR is dependent on cross-talk between phosphorylation and methylation events in the regulatory insertion (RI) element of the protein. Manipulating these posttranslational modifications (PTMs) prevented the maturation of wild-type CFTR and instead induced its degradation by ER quality control systems. Deletion of Phe508 (ΔF508), the most prevalent mutation in CF, and other mutations in CFTR that impair its trafficking, such as N1303K, also led to quantitative and qualitative PTM changes that prevented the maturation of misfolded CFTR. Further analysis revealed that a wild-type CFTR–like PTM pattern and function was restored in ΔF508 CFTR when cells were cultured at 28°C but only in the presence of the kinase CK2α. Furthermore, the ability to replicate this PTM pattern predicted the efficacy of treatments in restoring ΔF508 CFTR activity. Accordingly, evaluation of patient information revealed that point mutations of several of the modification sites are associated with clinical CF. These findings identify a minimal quantitative and qualitative PTM code for CFTR maturation that distinguishes correctly folded from misfolded CFTR.

    更新日期:2019-01-02
  • GPR35 promotes glycolysis, proliferation, and oncogenic signaling by engaging with the sodium potassium pump
    Sci. Signal. (IF 6.378) Pub Date : 2019-01-01
    Georg Schneditz, Joshua E. Elias, Ester Pagano, M. Zaeem Cader, Svetlana Saveljeva, Kathleen Long, Subhankar Mukhopadhyay, Maryam Arasteh, Trevor D. Lawley, Gordon Dougan, Andrew Bassett, Tom H. Karlsen, Arthur Kaser, Nicole C. Kaneider

    The sodium potassium pump (Na/K-ATPase) ensures the electrochemical gradient of a cell through an energy-dependent process that consumes about one-third of regenerated ATP. We report that the G protein–coupled receptor GPR35 interacted with the α chain of Na/K-ATPase and promotes its ion transport and Src signaling activity in a ligand-independent manner. Deletion of Gpr35 increased baseline Ca2+ to maximal levels and reduced Src activation and overall metabolic activity in macrophages and intestinal epithelial cells (IECs). In contrast, a common T108M polymorphism in GPR35 was hypermorphic and had the opposite effects to Gpr35 deletion on Src activation and metabolic activity. The T108M polymorphism is associated with ulcerative colitis and primary sclerosing cholangitis, inflammatory diseases with a high cancer risk. GPR35 promoted homeostatic IEC turnover, whereas Gpr35 deletion or inhibition by a selective pepducin prevented inflammation-associated and spontaneous intestinal tumorigenesis in mice. Thus, GPR35 acts as a central signaling and metabolic pacesetter, which reveals an unexpected role of Na/K-ATPase in macrophage and IEC biology.

    更新日期:2019-01-02
  • All three IP3 receptor isoforms generate Ca2+ puffs that display similar characteristics
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-18
    Jeffrey T. Lock, Kamil J. Alzayady, David I. Yule, Ian Parker

    Inositol 1,4,5-trisphosphate (IP3) evokes Ca2+ release through IP3 receptors (IP3Rs) to generate both local Ca2+ puffs arising from concerted openings of clustered IP3Rs and cell-wide Ca2+ waves. Imaging Ca2+ puffs with single-channel resolution yields information on the localization and properties of native IP3Rs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IP3Rs. Here, we used TIRF and light-sheet microscopy to image Ca2+ puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IP3R type 1, 2, or 3. Photorelease of the IP3 analog i-IP3 in all three cell lines evoked puffs with largely similar mean amplitudes, temporal characteristics, and spatial extents. Moreover, the single-channel Ca2+ flux was similar among isoforms, indicating that clusters of different IP3R isoforms contain comparable numbers of active channels. Our results show that all three IP3R isoforms cluster to generate local Ca2+ puffs and, contrary to findings of divergent properties from in vitro electrophysiological studies, display similar conductances and gating kinetics in intact cells.

    更新日期:2018-12-19
  • ORAI1, STIM1/2, and RYR1 shape subsecond Ca2+ microdomains upon T cell activation
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-18
    Björn-Philipp Diercks, René Werner, Paula Weidemüller, Frederik Czarniak, Lola Hernandez, Cari Lehmann, Annette Rosche, Aileen Krüger, Ulrike Kaufmann, Martin Vaeth, Antonio V. Failla, Bernd Zobiak, Farid I. Kandil, Daniel Schetelig, Alexandra Ruthenbeck, Chris Meier, Dmitri Lodygin, Alexander Flügel, Dejian Ren, Insa M. A. Wolf, Stefan Feske, Andreas H. Guse

    The earliest intracellular signals that occur after T cell activation are local, subsecond Ca2+ microdomains. Here, we identified a Ca2+ entry component involved in Ca2+ microdomain formation in both unstimulated and stimulated T cells. In unstimulated T cells, spontaneously generated small Ca2+ microdomains required ORAI1, STIM1, and STIM2. Super-resolution microscopy of unstimulated T cells identified a circular subplasmalemmal region with a diameter of about 300 nm with preformed patches of colocalized ORAI1, ryanodine receptors (RYRs), and STIM1. Preformed complexes of STIM1 and ORAI1 in unstimulated cells were confirmed by coimmunoprecipitation and Förster resonance energy transfer studies. Furthermore, within the first second after T cell receptor (TCR) stimulation, the number of Ca2+ microdomains increased in the subplasmalemmal space, an effect that required ORAI1, STIM2, RYR1, and the Ca2+ mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate). These results indicate that preformed clusters of STIM and ORAI1 enable local Ca2+ entry events in unstimulated cells. Upon TCR activation, NAADP-evoked Ca2+ release through RYR1, in coordination with Ca2+ entry through ORAI1 and STIM, rapidly increases the number of Ca2+ microdomains, thereby initiating spread of Ca2+ signals deeper into the cytoplasm to promote full T cell activation.

    更新日期:2018-12-19
  • CD45 exclusion– and cross-linking–based receptor signaling together broaden FcεRI reactivity
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-18
    James H. Felce, Erdinc Sezgin, Madina Wane, Heather Brouwer, Michael L. Dustin, Christian Eggeling, Simon J. Davis

    For many years, the high-affinity receptor for immunoglobulin E (IgE) FcεRI, which is expressed by mast cells and basophils, has been widely held to be the exemplar of cross-linking (that is, aggregation dependent) signaling receptors. We found, however, that FcεRI signaling could occur in the presence or absence of receptor cross-linking. Using both cell and cell-free systems, we showed that FcεRI signaling was stimulated by surface-associated monovalent ligands through the passive, size-dependent exclusion of the receptor-type tyrosine phosphatase CD45 from plasma membrane regions of FcεRI-ligand engagement. Similarly to the T cell receptor, FcεRI signaling could also be initiated in a ligand-independent manner. These data suggest that a simple mechanism of CD45 exclusion–based receptor triggering could function together with cross-linking–based FcεRI signaling, broadening mast cell and basophil reactivity by enabling these cells to respond to both multivalent and surface-presented monovalent antigens. These findings also strengthen the case that a size-dependent, phosphatase exclusion–based receptor triggering mechanism might serve generally to facilitate signaling by noncatalytic immune receptors.

    更新日期:2018-12-19
  • 5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein–coupled receptor D
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-18
    Tereza Bautzova, James R. F. Hockley, Teresa Perez-Berezo, Julien Pujo, Michael M. Tranter, Cleo Desormeaux, Maria Raffaella Barbaro, Lilian Basso, Pauline Le Faouder, Corinne Rolland, Pascale Malapert, Aziz Moqrich, Helene Eutamene, Alexandre Denadai-Souza, Nathalie Vergnolle, Ewan St John Smith, David I. Hughes, Giovanni Barbara, Gilles Dietrich, David C. Bulmer, Nicolas Cenac

    Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca2+ imaging of dorsal root ganglion (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)–positive DRG neurons through a phospholipase C (PLC)– and pertussis toxin–dependent mechanism, suggesting that the effect was mediated by a G protein–coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.

    更新日期:2018-12-19
  • Controlling chemokine secretion
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-18
    Erin R. Williams

    Activating the ion channel activity of TRPML2 on endosomes promotes cytokine secretion by macrophages.

    更新日期:2018-12-19
  • Spatially structured cell populations process multiple sensory signals in parallel in intact vascular endothelium
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-18
    Matthew D. Lee, Calum Wilson, Christopher D. Saunter, Charles Kennedy, John M. Girkin, John G. McCarron

    Blood flow, blood clotting, angiogenesis, vascular permeability, and vascular remodeling are each controlled by a large number of variable, noisy, and interacting chemical inputs to the vascular endothelium. The endothelium processes the entirety of the chemical composition to which the cardiovascular system is exposed, carrying out sophisticated computations that determine physiological output. Processing this enormous quantity of information is a major challenge facing the endothelium. We analyzed the responses of hundreds of endothelial cells to carbachol (CCh) and adenosine triphosphate (ATP) and found that the endothelium segregates the responses to these two distinct components of the chemical environment into separate streams of complementary information that are processed in parallel. Sensitivities to CCh and ATP mapped to different clusters of cells, and each agonist generated distinct signal patterns. The distinct signals were features of agonist activation rather than properties of the cells themselves. When there was more than one stimulus present, the cells communicated and combined inputs to generate new distinct signals that were nonlinear combinations of the inputs. Our results demonstrate that the endothelium is a structured, collaborative sensory network that simplifies the complex environment using separate cell clusters that are sensitive to distinct aspects of the overall biochemical environment and interactively compute signals from diverse but interrelated chemical inputs. These features enable the endothelium to selectively process separate signals and perform multiple computations in an environment that is noisy and variable.

    更新日期:2018-12-19
  • Restricting mitochondrial GRK2 post-ischemia confers cardioprotection by reducing myocyte death and maintaining glucose oxidation
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-11
    Priscila Y. Sato, J. Kurt Chuprun, Laurel A. Grisanti, Meryl C. Woodall, Brett R. Brown, Rajika Roy, Christopher J. Traynham, Jessica Ibetti, Anna M. Lucchese, Ancai Yuan, Konstantinos Drosatos, Doug G. Tilley, Erhe Gao, Walter J. Koch

    Increased abundance of GRK2 [G protein–coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.

    更新日期:2018-12-12
  • The β4 subunit of Cav1.2 channels is required for an optimal interferon response in cardiac muscle cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-11
    Eshwar R. Tammineni, Elba D. Carrillo, Rubén Soto-Acosta, Antonio H. Angel-Ambrocio, María C. García, Patricia Bautista-Carbajal, Rosa M. del Angel, Jorge A. Sánchez

    The auxiliary β4 subunit of the cardiac Cav1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the β4 subunit in H9c2 rat cardiac cells on the abundances of Ifnb mRNA [which encodes interferon-β (IFN-β)] and of the IFN-β–related genes Ddx58, Ifitm3, Irf7, Stat2, Ifih1, and Mx1, as well as on the abundances of the antiviral proteins DDX58, IRF7, STAT2, and IFITM3. Knocking down the β4 subunit in H9c2 cells reduced the expression of IFN-β–stimulated genes. In response to inhibition of the kinase JAK1, the abundances of β4 subunit mRNA and protein were decreased. β4 subunit abundance was increased, and it translocated to the nucleus, in cells treated with IFN-β, infected with dengue virus (DENV), or transfected with poly(I:C), a synthetic analog of double-stranded RNA. Cells that surrounded the virus-infected cells showed translocation of β4 subunit proteins to nuclei in response to spreading infection. We showed that the β4 subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter–driven reporter was increased in cells overexpressing the β4 subunit. Last, overexpressing β4 in undifferentiated and differentiated H9c2 cells reduced DENV infection and decreased the abundance of the viral proteins NS1, NS3, and E-protein. DENV infection and poly(I:C) also increased the concentration of intracellular Ca2+ in these cells. These findings suggest that the β4 subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection.

    更新日期:2018-12-12
  • Targeting PD-1
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-11
    John F. Foley

    Loss of an E3 ubiquitin ligase that targets PD-1 for degradation leads to inefficient antitumor responses by T cells.

    更新日期:2018-12-12
  • Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-11
    Nathan H. Roy, Joanna L. MacKay, Tanner F. Robertson, Daniel A. Hammer, Janis K. Burkhardt

    T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on “outside-in” signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function–associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling–induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

    更新日期:2018-12-12
  • The microRNA miR-7a-5p ameliorates ischemic brain damage by repressing α-synuclein
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-11
    TaeHee Kim, Suresh L. Mehta, Kahlilia C. Morris-Blanco, Anil K. Chokkalla, Bharath Chelluboina, Mary Lopez, Ruth Sullivan, Hung Tae Kim, Thomas D. Cook, Joo Yong Kim, HwuiWon Kim, Chanul Kim, Raghu Vemuganti

    Ischemic stroke, which is caused by a clot that blocks blood flow to the brain, can be severely disabling and sometimes fatal. We previously showed that transient focal ischemia in a rat model induces extensive temporal changes in the expression of cerebral microRNAs, with a sustained decrease in the abundance of miR-7a-5p (miR-7). Here, we evaluated the therapeutic efficacy of a miR-7 mimic oligonucleotide after cerebral ischemia in rodents according to the Stroke Treatment Academic Industry Roundtable (STAIR) criteria. Rodents were injected locally or systemically with miR-7 mimic before or after transient middle cerebral artery occlusion. Decreased miR-7 expression was observed in both young and aged rats of both sexes after cerebral ischemia. Pre- or postischemic treatment with miR-7 mimic decreased the lesion volume in both sexes and ages studied. Furthermore, systemic injection of miR-7 mimic into mice at 30 min (but not 2 hours) after cerebral ischemia substantially decreased the lesion volume and improved motor and cognitive functional recovery with minimal peripheral toxicity. The miR-7 mimic treatment substantially reduced the postischemic induction of α-synuclein (α-Syn), a protein that induces mitochondrial fragmentation, oxidative stress, and autophagy that promote neuronal cell death. Deletion of the gene encoding α-Syn abolished miR-7 mimic–dependent neuroprotection and functional recovery in young male mice. Further analysis confirmed that the transcript encoding α-Syn was bound and repressed by miR-7. Our findings suggest that miR-7 mimics may therapeutically minimize stroke-induced brain damage and disability.

    更新日期:2018-12-12
  • Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-04
    Yoon Namkung, Christian LeGouill, Sahil Kumar, Yubo Cao, Larissa B. Teixeira, Viktoriya Lukasheva, Jenna Giubilaro, Sarah C. Simões, Jean-Michel Longpré, Dominic Devost, Terence E. Hébert, Graciela Piñeyro, Richard Leduc, Claudio M. Costa-Neto, Michel Bouvier, Stéphane A. Laporte

    G protein–coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

    更新日期:2018-12-05
  • ERAD suppresses the starvation response
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-04
    Annalisa M. VanHook

    Endoplasmic reticulum–associated protein degradation suppresses the production of the fasting-associated hepatokine Fgf21.

    更新日期:2018-12-05
  • A division of labor in mTORC1 signaling and autophagy
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-04
    Sascha Martens

    In human cells, the p62 protein acts as an adaptor in various signaling pathways as well as a receptor for selective autophagy. In this issue of Science Signaling, Sanchez-Garrido et al. show that proteolytic cleavage of p62 by caspase-8 determines whether p62 functions as an mTORC1 signaling adaptor or autophagy receptor.

    更新日期:2018-12-05
  • FZD5 is a Gαq-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-04
    Shane C. Wright, Maria Consuelo Alonso Cañizal, Tobias Benkel, Katharina Simon, Christian Le Gouill, Pierre Matricon, Yoon Namkung, Viktoria Lukasheva, Gabriele M. König, Stéphane A. Laporte, Jens Carlsson, Evi Kostenis, Michel Bouvier, Gunnar Schulte, Carsten Hoffmann

    Frizzleds (FZDs) are a group of seven transmembrane–spanning (7TM) receptors that belong to class F of the G protein–coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the β-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)–based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.

    更新日期:2018-12-05
  • Regulated proteolysis of p62/SQSTM1 enables differential control of autophagy and nutrient sensing
    Sci. Signal. (IF 6.378) Pub Date : 2018-12-04
    Julia Sanchez-Garrido, Vanessa Sancho-Shimizu, Avinash R. Shenoy

    The multidomain scaffold protein p62 (also called sequestosome-1) is involved in autophagy, antimicrobial immunity, and oncogenesis. Mutations in SQSTM1, which encodes p62, are linked to hereditary inflammatory conditions such as Paget’s disease of the bone, frontotemporal dementia (FTD), amyotrophic lateral sclerosis, and distal myopathy with rimmed vacuoles. Here, we report that p62 was proteolytically trimmed by the protease caspase-8 into a stable protein, which we called p62TRM. We found that p62TRM, but not full-length p62, was involved in nutrient sensing and homeostasis through the mechanistic target of rapamycin complex 1 (mTORC1). The kinase RIPK1 and caspase-8 controlled p62TRM production and thus promoted mTORC1 signaling. An FTD-linked p62 D329G polymorphism and a rare D329H variant could not be proteolyzed by caspase-8, and these noncleavable variants failed to activate mTORC1, thereby revealing the detrimental effect of these mutations. These findings on the role of p62TRM provide new insights into SQSTM1-linked diseases and mTORC1 signaling.

    更新日期:2018-12-05
  • Priming the liver for a feast after famine
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-27
    Wei Wong

    Food perception activates a hypothalamus-to-liver pathway that prepares the liver for nutrient influx.

    更新日期:2018-11-28
  • Thinking through acidic Ca2+ stores
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-27
    Sandip Patel, Eugen Brailoiu

    Glutamate signaling regulates neuronal activity and synaptic plasticity, which underlies learning and memory. In this issue of Science Signaling, Foster et al. found that metabotropic glutamate receptors mediate long-term potentiation in hippocampal neurons by mobilizing acidic endolysosomal Ca2+ stores through the intracellular messenger NAADP.

    更新日期:2018-11-28
  • β-Barrel outer membrane proteins suppress mTORC2 activation and induce autophagic responses
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-27
    Anu Chaudhary, Cassandra Kamischke, Mara Leite, Melissa A. Altura, Loren Kinman, Hemantha Kulasekara, Marie-Pierre Blanc, Guoxing Wang, Cox Terhorst, Samuel I. Miller

    The outer membranes of Gram-negative bacteria and mitochondria contain proteins with a distinct β-barrel tertiary structure that could function as a molecular pattern recognized by the innate immune system. Here, we report that purified outer membrane proteins (OMPs) from different bacterial and mitochondrial sources triggered the induction of autophagy-related endosomal acidification, LC3B lipidation, and p62 degradation. Furthermore, OMPs reduced the phosphorylation and therefore activation of the multiprotein complex mTORC2 and its substrate Akt in macrophages and epithelial cells. The cell surface receptor SlamF8 and the DNA-protein kinase subunit XRCC6 were required for these OMP-specific responses in macrophages and epithelial cells, respectively. The addition of OMPs to mouse bone marrow–derived macrophages infected with Salmonella Typhimurium facilitated bacterial clearance. These data identify a specific cellular response mediated by bacterial and mitochondrial OMPs that can alter inflammatory responses and influence the killing of pathogens.

    更新日期:2018-11-28
  • Aspirin ameliorates experimental autoimmune encephalomyelitis through interleukin-11–mediated protection of regulatory T cells
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-27
    Susanta Mondal, Malabendu Jana, Sridevi Dasarathi, Avik Roy, Kalipada Pahan

    Multiple sclerosis (MS) is a human disease that results from autoimmune T cells targeting myelin protein that is expressed within the central nervous system. In MS, the number of FoxP3-expressing regulatory T cells (Tregs) is reduced, which facilitates the activation of autoreactive T cells. Because aspirin (acetylsalicylic acid) is the most widely used nonsteroidal anti-inflammatory drug, we examined its immunomodulatory effect in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We found that low-dose aspirin suppressed the clinical symptoms of EAE in mouse models of both relapsing-remitting and chronic disease. Aspirin reduced the development of EAE driven by myelin basic protein (MBP)–specific T cells and the associated perivascular cuffing, inflammation, and demyelination. The effects of aspirin required the presence of CD25+FoxP3+ Tregs. Aspirin increased the amounts of Foxp3 and interleukin-4 (IL-4) in T cells and suppressed the differentiation of naïve T cells into T helper 17 (TH17) and TH1 cells. Aspirin also increased the transcription of Il11 mediated by the transcription factor CREB, which was necessary for the generation of Tregs. Neutralization of IL-11 negated the effects of aspirin on Treg development and exacerbated EAE. Furthermore, we found that IL-11 alone was sufficient to maintain the percentage of FoxP3+ Tregs and protect mice from EAE. These results identify a previously uncharacterized mode of action of aspirin.

    更新日期:2018-11-28
  • Hippocampal mGluR1-dependent long-term potentiation requires NAADP-mediated acidic store Ca2+ signaling
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-27
    William J. Foster, Henry B. C. Taylor, Zahid Padamsey, Alexander F. Jeans, Antony Galione, Nigel J. Emptage

    Acidic organelles, such as endosomes and lysosomes, store Ca2+ that is released in response to intracellular increases in the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). In neurons, NAADP and Ca2+ signaling contribute to synaptic plasticity, a process of activity-dependent long-term potentiation (LTP) [or, alternatively, long-term depression (LTD)] of synaptic strength and neuronal transmission that is critical for neuronal function and memory formation. We explored the function of and mechanisms regulating acidic Ca2+ store signaling in murine hippocampal neurons. We found that metabotropic glutamate receptor 1 (mGluR1) was coupled to NAADP signaling that elicited Ca2+ release from acidic stores. In turn, this released Ca2+-mediated mGluR1-dependent LTP by transiently inhibiting SK-type K+ channels, possibly through the activation of protein phosphatase 2A. Genetically removing two-pore channels (TPCs), which are endolysosomal-specific ion channels, switched the polarity of plasticity from LTP to LTD, indicating the importance of specific receptor store coupling and providing mechanistic insight into how mGluR1 can produce both synaptic potentiation and synaptic depression.

    更新日期:2018-11-28
  • The two faces of cGAS in cancer and immunity
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-20
    Leslie K. Ferrarelli

    Cytosolic DNA sensing by cGAS promotes immunity, but its nuclear localization can promote tumorigenesis.

    更新日期:2018-11-21
  • Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-20
    Jimmy Van den Eynden, Ganesh Umapathy, Arghavan Ashouri, Diana Cervantes-Madrid, Joanna Szydzik, Kristina Ruuth, Jan Koster, Erik Larsson, Jikui Guan, Ruth H. Palmer, Bengt Hallberg

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is a clinical target of major interest in cancer. Mutations and rearrangements in ALK trigger the activation of the encoded receptor and its downstream signaling pathways. ALK mutations have been identified in both familial and sporadic neuroblastoma cases as well as in 30 to 40% of relapses, which makes ALK a bona fide target in neuroblastoma therapy. Tyrosine kinase inhibitors (TKIs) that target ALK are currently in clinical use for the treatment of patients with ALK-positive non–small cell lung cancer. However, monotherapy with the ALK inhibitor crizotinib has been less encouraging in neuroblastoma patients with ALK alterations, raising the question of whether combinatorial therapy would be more effective. In this study, we established both phosphoproteomic and gene expression profiles of ALK activity in neuroblastoma cells exposed to first- and third-generation ALK TKIs, to identify the underlying molecular mechanisms and identify relevant biomarkers, signaling networks, and new therapeutic targets. This analysis has unveiled various important leads for novel combinatorial treatment strategies for patients with neuroblastoma and an increased understanding of ALK signaling involved in this disease.

    更新日期:2018-11-21
  • The HDAC3–SMARCA4–miR-27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-20
    Narendra Bharathy, Noah E. Berlow, Eric Wang, Jinu Abraham, Teagan P. Settelmeyer, Jody E. Hooper, Matthew N. Svalina, Yoshihiro Ishikawa, Keith Zientek, Zia Bajwa, Martin W. Goros, Brian S. Hernandez, Johannes E. Wolff, Michelle A. Rudek, Linping Xu, Nicole M. Anders, Ranadip Pal, Alexandria P. Harrold, Angela M. Davies, Arya Ashok, Darnell Bushby, Maria Mancini, Christopher Noakes, Neal C. Goodwin, Peter Ordentlich, James Keck, Douglas S. Hawkins, Erin R. Rudzinski, Bishwanath Chatterjee, Hans Peter Bächinger, Frederic G. Barr, Jennifer Liddle, Benjamin A. Garcia, Atiya Mansoor, Theodore J. Perkins, Christopher R. Vakoc, Joel E. Michalek, Charles Keller

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I–specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3–SMARCA4–miR-27a–PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.

    更新日期:2018-11-21
  • Integrated proximal proteomics reveals IRS2 as a determinant of cell survival in ALK-driven neuroblastoma
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-20
    Kristina B. Emdal, Anna-Kathrine Pedersen, Dorte B. Bekker-Jensen, Alicia Lundby, Shana Claeys, Katleen De Preter, Frank Speleman, Chiara Francavilla, Jesper V. Olsen

    Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry–based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI–sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis.

    更新日期:2018-11-21
  • IFN-γ–inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-20
    Diana Saleiro, Gavin T. Blyth, Ewa M. Kosciuczuk, Patrick A. Ozark, Beata Majchrzak-Kita, Ahmet D. Arslan, Mariafausta Fischietti, Neha K. Reddy, Curt M. Horvath, Roger J. Davis, Eleanor N. Fish, Leonidas C. Platanias

    It is well established that activation of the transcription factor signal transducer and activator of transcription 1 (STAT1) is required for the interferon-γ (IFN-γ)–mediated antiviral response. Here, we found that IFN-γ receptor stimulation also activated Unc-51–like kinase 1 (ULK1), an initiator of Beclin-1–mediated autophagy. Furthermore, the interaction between ULK1 and the mitogen-activated protein kinase kinase kinase MLK3 (mixed lineage kinase 3) was necessary for MLK3 phosphorylation and downstream activation of the kinase ERK5. This autophagy-independent activity of ULK1 promoted the transcription of key antiviral IFN-stimulated genes (ISGs) and was essential for IFN-γ–dependent antiviral effects. These findings define a previously unknown IFN-γ pathway that appears to be a key element of the antiviral response.

    更新日期:2018-11-21
  • Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein and ubiquitin ligase MDM2
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    JingJing Liu, Xianlin Zou, Tetsuya Gotoh, Anne M. Brown, Liang Jiang, Esther L. Wisdom, Jae Kyoung Kim, Carla V. Finkielstein

    The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by β-transducin repeat–containing proteins (β-TrCPs), which are F-box–containing proteins that function as substrate recognition subunits of the SCFβ-TRCP ubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

    更新日期:2018-11-14
  • The basics of mechanotransduction
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Erin R. Williams

    Basic residues in the transmembrane domain of the T cell receptor α chain promote its association with CD3 signaling chains.

    更新日期:2018-11-14
  • A cytoskeletal anchor connects ischemic mitochondrial fission to myocardial senescence
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Michael J. Boyer, Satoru Eguchi

    The interplay between the actin cytoskeleton and mitochondria has been implicated in cell and tissue homeostasis and physiological function. In this issue of Science Signaling, Nishimura et al. demonstrate that inhibiting the interaction of filamin A, an actin cytoskeleton regulator, with Drp1, a modulator of mitochondrial dynamics, attenuates mitochondrial hyperfission and cardiomyocyte senescence after myocardial infarction.

    更新日期:2018-11-14
  • The transmembrane adaptor protein NTAL limits mast cell chemotaxis toward prostaglandin E2
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Ivana Halova, Monika Bambouskova, Lubica Draberova, Viktor Bugajev, Petr Draber

    Chemotaxis of mast cells is one of the crucial steps in their development and function. Non–T cell activation linker (NTAL) is a transmembrane adaptor protein that inhibits the activation of mast cells and B cells in a phosphorylation-dependent manner. Here, we studied the role of NTAL in the migration of mouse mast cells stimulated by prostaglandin E2 (PGE2). Although PGE2 does not induce the tyrosine phosphorylation of NTAL, unlike IgE immune complex antigens, we found that loss of NTAL increased the chemotaxis of mast cells toward PGE2. Stimulation of mast cells that lacked NTAL with PGE2 enhanced the phosphorylation of AKT and the production of phosphatidylinositol 3,4,5-trisphosphate. In resting NTAL-deficient mast cells, phosphorylation of an inhibitory threonine in ERM family proteins accompanied increased activation of β1-containing integrins, which are features often associated with increased invasiveness in tumors. Rescue experiments indicated that only full-length, wild-type NTAL restored the chemotaxis of NTAL-deficient cells toward PGE2. Together, these data suggest that NTAL is a key inhibitor of mast cell chemotaxis toward PGE2, which may act through the RHOA/ERM/β1-integrin and PI3K/AKT axes.

    更新日期:2018-11-14
  • Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission–associated myocardial senescence
    Sci. Signal. (IF 6.378) Pub Date : 2018-11-13
    Akiyuki Nishimura, Tsukasa Shimauchi, Tomohiro Tanaka, Kakeru Shimoda, Takashi Toyama, Naoyuki Kitajima, Tatsuya Ishikawa, Naoya Shindo, Takuro Numaga-Tomita, Satoshi Yasuda, Yoji Sato, Koichiro Kuwahara, Yoshito Kumagai, Takaaki Akaike, Tomomi Ide, Akio Ojida, Yasuo Mori, Motohiro Nishida

    Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission–accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission–associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding–dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1–filamin A interaction by cilnidipine suppressed mitochondrial hyperfission–associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development.

    更新日期:2018-11-14
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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