Signaling Off Research Articles

Differentially Expressed Candidate miRNAs of Day 16 Bovine Embryos on the Regulation of Pregnancy Establishment in Dairy Cows.

Recent advances in high-throughput in silico techniques translate experimental data into meaningful biological networks through which the role of individual proteins, interactions, and their biological functions are comprehended. The study objective was to identify differentially expressed (DE) miRNAs between the day 16 competent, elongated embryo from normal cows and the day 16 noncompetent, tubular embryos from repeat breeder cows, assimilate DE-miRNAs to their target genes, and group target genes based on biological function using in silico methods. The 84 prioritized bovine-specific miRNAs were investigated by RT-PCR, and the results showed that 19 were differentially expressed (11 up- and 8 down-regulated) in the competent embryos compared to noncompetent ones (p ≤ 0.05; fold regulation ≥ 2 magnitudes). Top-ranked integrated genes of DE-miRNAs predicted various biological and molecular functions, cellular processes, and signaling pathways. Further, analysis of the categorized groups of genes showed association with signaling pathways, turning on or off key genes and transcription factors regulating the development of embryo, placenta, and various organs. In conclusion, highly DE-miRNAs in day 16 bovine conceptus regulated the embryogenesis and pregnancy establishment. The elucidated miRNA-mRNA interactions in this study were mostly based on predictions from public databases. Therefore, the causal regulations of these interactions and mechanisms require further functional characterization.

Glycyrrhizin prevents 3-nitropropionic acid-induced neurotoxicity by downregulating HMGB1/TLR4/NF-κB p65 signaling, and attenuating oxidative stress, inflammation, and apoptosis in rats

AimsGlycyrrhizin (Glyc) is a saponin triterpenoid that has signified its efficacy against Huntington's disease (HD). Nonetheless, its mechanism has not been fully clarified. Accordingly, this study was designed to evaluate the plausible mechanism of action of Glyc against 3-nitropropionic acid (3-NP)-induced HD. Main methodsRats were treated with Glyc (50 mg/kg, i.p.) for 3 weeks and 3-NP (10 mg/kg, i.p.) was administered at the latter 2 weeks alongside to induce HD. Key findingsAnimals exposed to 3-NP revealed a reduction in body weight, neurobehavioral abnormalities, and various deleterious effects related to overexpression of HMGB1 such as oxidative stress, apoptosis, and inflammation. Promisingly, Glyc administration provided valuable effects by reversing the decline in body weight with improved neurobehavioral deficits. Ameliorating oxidative stress via restoring GSH, SOD, and Nrf2 alongside with MDA suppression was evident. Furthermore, Glyc switched the HMGB1/TLR4/NF-κB p65 signaling off, reduced IL-6, IL-β, TNF-α, caspase-3, and increased Bcl-2 as well as BDNF. All these beneficial effects were mirrored by a better histopathological picture upon using Glyc that suppressed gliosis by reducing GFAP expression as observed in the immunohistochemistry results. SignificanceAccordingly, the current study demonstrated a promising neuroprotective effect of Glyc against experimentally induced HD through alleviating deleterious events by diverse mechanisms.

Leptospira interrogans Aer2: an Unusual Membrane-Bound PAS-Heme Oxygen Sensor.

In this study, we provide the first characterization of a chemoreceptor from Leptospira interrogans, the cause of leptospirosis. This receptor is related to the Aer2 receptors that have been studied in other bacteria. In those organisms, Aer2 is a soluble receptor with one or two PAS-heme domains and signals in response to O2 binding. In contrast, L. interrogans Aer2 (LiAer2) is an unusual membrane-bound Aer2 with a periplasmic domain and three cytoplasmic PAS-heme domains. Each of the three PAS domains bound b-type heme via conserved Eη-His residues. They also bound O2 and CO with similar affinities to each other and other PAS-heme domains. However, all three PAS domains were uniquely hexacoordinate in the deoxy-heme state, whereas other Aer2-PAS domains are pentacoordinate. Similar to other Aer2 receptors, LiAer2 could hijack the E. coli chemotaxis pathway but only when it was expressed with an E. coli high-abundance chemoreceptor. Unexpectedly, the response was inverted relative to classic Aer2 receptors. That is, LiAer2 caused E. coli to tumble (it was signal-on) in the absence of O2 and to stop tumbling in its presence. Thus, an endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2, and its displacement for gas-binding turned signaling off. This response also occurred in a soluble version of LiAer2 lacking the periplasmic domain, transmembrane (TM) region, and first two PAS domains, meaning that PAS3 alone was sufficient for O2-mediated control. Future studies are needed to understand the unique signaling mechanisms of this unusual Aer2 receptor. IMPORTANCE Leptospira interrogans, the cause of the zoonotic infection leptospirosis, is found in soil and water contaminated with animal urine. L. interrogans survives in complex environments with the aid of 12 chemoreceptors, none of which has been explicitly studied. In this study, we characterized the first L. interrogans chemoreceptor, LiAer2, and reported its unique characteristics. LiAer2 is membrane-bound, has three cytoplasmic PAS-heme domains that each bound hexacoordinate b-type heme and O2 turned LiAer2 signaling off. An endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2 and its displacement for O2-binding turned signaling off. Our study corroborated previous findings that Aer2 receptors are O2 sensors, but also demonstrated that they do not all function the same way.

Epigenetic Modulation of SPCA2 Reverses Epithelial to Mesenchymal Transition in Breast Cancer Cells.

Simple SummaryThe triple receptor negative breast cancer subtype, or TNBC, currently has no tailored treatment options. TNBC is highly metastatic, associated with high patient mortality, and disproportionately occurs in Black/African American women where it contributes to racial disparities in health outcomes. Therefore, we focused on new therapeutic approaches to TNBC. We discovered that levels of the Calcium-ATPase SPCA2 are abnormally low in TNBC and that these low levels correlate with poor survival prognosis in patients. Previously, we showed that recombinant SPCA2 prevented TNBC cells from acquiring aggressive “mesenchymal” properties associated with metastasis both in vitro and in vivo. These findings motivated us to search for drugs that turn the SPCA2 gene back on in TNBC cells. In this study, we show that histone deacetylase inhibitors increase SPCA2 levels, activate Ca2+ signaling and convert cancer cells to a less aggressive “epithelial” state. These findings could lead to new treatment options for TNBC.The secretory pathway Ca2+-ATPase SPCA2 is a tumor suppressor in triple receptor negative breast cancer (TNBC), a highly aggressive molecular subtype that lacks tailored treatment options. Low expression of SPCA2 in TNBC confers poor survival prognosis in patients. Previous work has established that re-introducing SPCA2 to TNBC cells restores basal Ca2+ signaling, represses mesenchymal gene expression, mitigates tumor migration in vitro and metastasis in vivo. In this study, we examined the effect of histone deacetylase inhibitors (HDACi) in TNBC cell lines. We show that the pan-HDACi vorinostat and the class I HDACi romidepsin induce dose-dependent upregulation of SPCA2 transcript with concurrent downregulation of mesenchymal markers and tumor cell migration characteristic of epithelial phenotype. Silencing SPCA2 abolished the ability of HDACi to reverse epithelial to mesenchymal transition (EMT). Independent of ATPase activity, SPCA2 elevated resting Ca2+ levels to activate downstream components of non-canonical Wnt/Ca2+ signaling. HDACi treatment led to SPCA2-dependent phosphorylation of CAMKII and β-catenin, turning Wnt signaling off. We conclude that SPCA2 mediates the efficacy of HDACi in reversing EMT in TNBC by a novel mode of non-canonical Wnt/Ca2+ signaling. Our findings provide incentive for screening epigenetic modulators that exploit Ca2+ signaling pathways to reverse EMT in breast tumors.

Feeling the heat: source-sink mismatch as a mechanism underlying the failure of thermal tolerance.

A mechanistic explanation for the tolerance limits of animals at high temperatures is still missing, but one potential target for thermal failure is the electrical signaling off cells and tissues. With this in mind, here I review the effects of high temperature on the electrical excitability of heart, muscle and nerves, and refine a hypothesis regarding high temperature-induced failure of electrical excitation and signal transfer [the temperature-dependent deterioration of electrical excitability (TDEE) hypothesis]. A central tenet of the hypothesis is temperature-dependent mismatch between the depolarizing ion current (i.e. source) of the signaling cell and the repolarizing ion current (i.e. sink) of the receiving cell, which prevents the generation of action potentials (APs) in the latter. A source-sink mismatch can develop in heart, muscles and nerves at high temperatures owing to opposite effects of temperature on source and sink currents. AP propagation is more likely to fail at the sites of structural discontinuities, including electrically coupled cells, synapses and branching points of nerves and muscle, which impose an increased demand of inward current. At these sites, temperature-induced source-sink mismatch can reduce AP frequency, resulting in low-pass filtering or a complete block of signal transmission. In principle, this hypothesis can explain a number of heat-induced effects, including reduced heart rate, reduced synaptic transmission between neurons and reduced impulse transfer from neurons to muscles. The hypothesis is equally valid for ectothermic and endothermic animals, and for both aquatic and terrestrial species. Importantly, the hypothesis is strictly mechanistic and lends itself to experimental falsification.

Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains.

The type III CRISPR–Cas systems provide immunity against invading nucleic acids through the coordinated transcription-dependent DNA targeting and cyclic adenylate (cAn)-activated RNA degradation. Here, we show that both these pathways contribute to the Streptococcus thermophilus (St) type III-A CRISPR–Cas immunity. HPLC-MS analysis revealed that in the heterologous Escherichia coli host the StCsm effector complex predominantly produces cA5 and cA6. cA6 acts as a signaling molecule that binds to the CARF domain of StCsm6 to activate non-specific RNA degradation by the HEPN domain. By dissecting StCsm6 domains we demonstrate that both CARF and HEPN domains act as ring nucleases that degrade cAns to switch signaling off. CARF ring nuclease converts cA6 to linear A6>p and to the final A3>p product. HEPN domain, which typically degrades RNA, also shows ring nuclease activity and indiscriminately degrades cA6 or other cAns down to A>p. We propose that concerted action of both ring nucleases enables self-regulation of the RNase activity in the HEPN domain and eliminates all cAn secondary messengers in the cell when viral infection is combated by a coordinated action of Csm effector and the cA6-activated Csm6 ribonuclease.

Abstract B27: Identification of uveal melanoma disseminated cancer cell dormancy mechanisms

Abstract Introduction: Uveal melanoma (UM) is the most common intraocular cancer. Most patients succumb to liver metastasis after long asymptomatic periods through unknown mechanisms. After primary tumor resection, UM disseminated cancer cells (DCCs) remain dormant more than 15 years before growing lethal liver metastases. We propose that UM DCC dormancy may be responsible for the time delay to liver metastasis observed in clinic, which may represent an opportunity to prevent UM recurrences. Methods: To characterize dormant and proliferative UM disseminated cells in the liver, we used an in vivo mouse model in which UM cells injected into the spleen disseminate and colonize the liver. In this model, UM DCCs stay dormant (up to 3 months) until some DCCs form proliferative metastasis. Liver DCCs that stayed dormant or formed metastasis were cell sorted and studied via gene transcription RNAseq. In addition, we monitored NR2F1, an orphan nuclear receptor of retinoic acid (RA) family, RA receptors (RARs) and RAR antagonist PRAME along with the YAP-TAZ pathway, which are linked to UM progression. Results: Our data indicate that RA signaling may play a critical role in UM DCCs switch to become dormant. RARs α, β, and γ mediate transcriptional regulation upon RA ligand binding and are critical for the correct balance between self-renewal and differentiation of tissue stem cells. RA is highly abundant in the liver tumor microenvironment (TME). We found that RA regulated genes such as RARβ and NR2F1 are upregulated in UM single cells (UMsc), which are low for proliferation markers (Ki67) compared to large metastases (UMm). Our data indicate that NR2F1 is growth suppressor in UM organoid cultures, and that RARβ and NR2F1 are key mediators in UM DCCs dormancy. We also observed a downregulation of PRAME, a poor-prognosis indicator in UM patients. UMsc were RARβhigh, NR2F1high, and PRAMElow; conversely, UMm were RARβlow, NR2F1low and PRAMEhigh. We propose that upon liver colonization those DCCs able to sense RA in the TME activate RA signaling and switch off growth while engaging dormancy (RARβhigh, NR2F1high, PRAMElow) pathways. In vitro knockdown of NR2F1 in UM cell line with Gαq/11 mutation leads to a decrease in dormancy markers and an increase in YAP downstream targets, indicating an interplay between NR2F1 and the Hippo signaling pathway. Importantly, the use of retinoids and 5-azacytidine or the use of Gαq/11 inhibitors on UM cells in organoid cultures was able to turn on a dormancy program associated with melanocytic or neuritic differentiation. Conclusion: Our data allow to conclude that UM DCCs dormancy may be linked to the activation of dormancy pathways including enhanced NR2F1 and RARβ signaling and the downregulation of pathways linked to poor prognosis (PRAMEhigh) and downstream signaling of Gαq/11 through YAP-TAZ transcriptional programs. Citation Format: Melisa Lopez-Anton, Eduardo F. Farias, Vivian Chua, Mizue Terai, Anna Rita Nobre, Philip Wedegaertner, Takami Sato, Jeffrey Benovic, Andrew Aplin, Julio Aguirre-Ghiso. Identification of uveal melanoma disseminated cancer cell dormancy mechanisms [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B27.

When More Is Less: Dual Phosphorylation Protects Signaling Off State against Overexpression

Kinases in signaling pathways are commonly activated by multisite phosphorylation. For example, the mitogen-activated protein kinase Erk is activated by its kinase Mek by two consecutive phosphorylations within its activation loop. In this article, we use kinetic models to study how the activation of Erk is coupled to its abundance. Intuitively, Erk activity should rise with increasing amounts of Erk protein. However, a mathematical model shows that the signaling off state is robust to increasing amounts of Erk, and Erk activity may even decline with increasing amounts of Erk. This counterintuitive, bell-shaped response of Erk activity to increasing amounts of Erk arises from the competition of the unmodified and single phosphorylated form of Erk for access to its kinase Mek. This shows that phosphorylation cycles can contain an intrinsic robustness mechanism that protects signaling from aberrant activation e.g., by gene expression noise or kinase overexpression after gene duplication events in diseases like cancer.

Osterix functions downstream of anti-Müllerian hormone signaling to regulate Müllerian duct regression

In mammals, the developing reproductive tract primordium of male and female fetuses consists of the Wolffian duct and the Müllerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct, and upper vagina. It is well established that transforming growth factor-β family member anti-Müllerian hormone (AMH) secreted from the fetal testis and its type 1 and 2 receptors expressed in MD mesenchyme regulate MD regression. However, little is known about the molecular network regulating downstream actions of AMH signaling. To identify potential AMH-induced genes and regulatory networks controlling MD regression in a global nonbiased manner, we examined transcriptome differences in MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) by RNA-seq analysis of purified fetal MD mesenchymal cells. This analysis found 82 genes up-regulated in males during MD regression and identified Osterix (Osx)/Sp7, a key transcriptional regulator of osteoblast differentiation and bone formation, as a downstream effector of AMH signaling during MD regression. Osx/OSX was expressed in a male-specific pattern in MD mesenchyme during MD regression. OSX expression was lost in mutant males without AMH signaling. In addition, transgenic mice ectopically expressing human AMH in females induced a male pattern of Osx expression. Together, these results indicate that AMH signaling is necessary and sufficient for Osx expression in the MD mesenchyme. In addition, MD regression was delayed in Osx-null males, identifying Osx as a factor that regulates MD regression.

Nitric Oxide Level Is Self-Regulating and Also Regulates Its ROS Partners.

Nitric oxide (NO) is a free radical recognized as an omnipresent inter- and intra-cellular signaling molecule involved in the regulation of an extraordinary range of diverse cellular functions in plants (Besson-Bard et al., 2008). All NO derivatives are called reactive nitrogen species (RNS) which include more favorable structures such as nitrosonium cation (NO+) and nitroxyl radical (NO−) as a result of NO gaining or losing electrons, and the products of the reaction between NO and its close partners, reactive oxygen species (ROS), such as peroxynitrite (ONOO−), and the NOx compounds (NO2, N2O3, and N2O4; Bellin et al., 2013). Although it is known that NO may regulate gene transcription and activate secondary messengers, the ways in which NO functions are still mostly unidentified (Besson-Bard et al., 2008; Palmieri et al., 2008; Gaupels et al., 2011). In the last decade, however, it has been shown that NO is also able to control different biological processes in plants by directly modifying proteins through covalent post-translational modifications (PTMs) giving rise to nitration, nitrosylation or S-nitrosylation (Romero-Puertas et al., 2013). S-nitrosylation is the covalent binding of a NO group to a cysteine residue and probably the better known NO-dependent PTM as more than 1000 proteins have been shown as targets of S-nitrosylation (Kovacs and Lindermayr, 2013), although the functional effect of this modification has only been analyzed in around 2% of these proteins (Astier et al., 2012; Kovacs and Lindermayr, 2013; Romero-Puertas et al., 2013). Nitration, in which 3-nitrotyrosine is produced after a nitrite group is added to the ortho-position of Tyr residues, is another NO-dependent PTM, although so far analyzed to a lesser degree than S-nitrosylation (N-Tyr; Vandelle and Delledonne, 2011). These PTMs are able to modify the activity, location, aggregation, or even stability of proteins (de Pinto et al., 2013; Gibbs et al., 2014; Albertos et al., 2015). We should bear in mind that NO function depends on the rate and location of its production and that NO level will determine a cytotoxic or stimulating effect (Beligni and Lamattina, 2001; Serrano et al., 2012). Thus, a precise control of NO level by switching the NO signaling off or not seems to be a crucial event for plant survival, and it appears that plants have developed many strategies to achieve it. Reactive oxygen species (ROS) comprises oxygen derivatives species produced by the reduction of oxygen, such as superoxide radicals (O2.-), hydroxyl radicals (·OH), peroxyl radicals (ROO·), and alkoxyl radicals (RO·) and also some non-radical compounds such as hydrogen peroxide (H2O2), the singlet oxygen (1O2), ozone (O3), and hypochlorous acid (HOCl−) (Halliwell and Gutteridge, 2007). Although research on ROS, which have strong oxidizing potential, initially focused on cytotoxicity, in recent years, it has become clear that they can also function as signaling molecules in most cellular processes (Baxter et al., 2014). Thus, plants have developed a means of utilizing lower concentrations of ROS as signaling molecules under certain physiological and stress conditions (Petrov and Van Breusegem, 2012). Genetic and pharmacological techniques have demonstrated that different ROS species can affect nuclear gene expression by responding to a variety of environmental stimuli (Sandalio and Romero-Puertas, 2015). However, a finely tuned balance between ROS scavenging and ROS production is necessary to determine their level and impact as damaging and signaling molecules (de Pinto et al., 2012; Baxter et al., 2014).

Overlapping and asymmetric functions of TCR signaling during thymic selection of CD4 and CD8 lineages.

TCR signaling plays a central role in directing developmental fates of thymocytes. Current models suggest TCR signal duration directs CD4 versus CD8 lineage development. To investigate the role of TCR signaling during positive selection directly, we switched signaling off in a cohort of selecting thymocytes and followed, in time, their subsequent fate. We did this using an inducible Zap70 transgenic mouse model that allowed Zap70-dependent signaling to be turned on and then off again. Surprisingly, loss of TCR signaling in CD4(+)CD8(lo) thymocytes did not prevent their development into committed CD4 single positives (SPs), nor their continued maturation to HSA(lo) SPs. However, numbers of CD4 SPs underwent a substantial decline following loss of Zap70 expression, suggesting an essential survival role for the kinase. Termination of TCR signaling is considered an essential step in CD8 lineage development. Loss of Zap70 expression, however, resulted in the rapid death of CD8 lineage precursor thymocytes and a failure to generate CD8 SPs. Significantly, extending the window of Zap70 expression was sufficient for generation and export of both CD4 and CD8 T cells. These data reveal a parallel requirement for TCR-mediated survival signaling, but an asymmetric requirement for TCR-mediated maturation signals.

Chemical and genetic exploration of jasmonate biosynthesis and signaling paths

Jasmonates are lipid-derived compounds that act as signals in plant stress responses and developmental processes. Enzymes participating in biosynthesis of jasmonic acid (JA) and components of JA signaling have been extensively characterized by biochemical and molecular-genetic tools. Mutants have helped to define the pathway for synthesis of jasmonoyl-L-isoleucine (JA-Ile), the bioactive form of JA, and to identify the F-box protein COI1 as central regulatory unit. Details on the molecular mechanism of JA signaling were recently unraveled by the discovery of JAZ proteins that together with the adaptor protein NINJA and the general co-repressor TOPLESS form a transcriptional repressor complex. The current model of JA perception and signaling implies the SCF(COI1) complex operating as E3 ubiquitin ligase that upon binding of JA-Ile targets JAZ proteins for degradation by the 26S proteasome pathway, thereby allowing MYC2 and other transcription factors to activate gene expression. Chemical strategies, as integral part of jasmonate research, have helped the establishment of structure-activity relationships and the discovery of (+)-7-iso-JA-L-Ile as the major bioactive form of the hormone. The transient nature of its accumulation highlights the need to understand catabolism and inactivation of JA-Ile and recent studies indicate that oxidation of JA-Ile by cytochrome P450 monooxygenase is the major mechanism for turning JA signaling off. Plants contain numerous JA metabolites, which may have pronounced and differential bioactivity. A major challenge in the field of plant lipid signaling is to identify the cognate receptors and modes of action of these bioactive jasmonates/oxylipins.

Defining Components of the ßcatenin Destruction Complex and Exploring Its Regulation and Mechanisms of Action during Development

BackgroundA subset of signaling pathways play exceptionally important roles in embryonic and post-embryonic development, and mis-regulation of these pathways occurs in most human cancers. One such pathway is the Wnt pathway. The primary mechanism keeping Wnt signaling off in the absence of ligand is regulated proteasomal destruction of the canonical Wnt effector ßcatenin (or its fly homolog Armadillo). A substantial body of evidence indicates that SCFβTrCP mediates βcat destruction, however, an essential role for Roc1 has not been demonstrated in this process, as would be predicted. In addition, other E3 ligases have also been proposed to destroy βcat, suggesting that βcat destruction may be regulated differently in different tissues.Methodology/Principal FindingsHere we used cultured Drosophila cells, human colon cancer cells, and Drosophila embryos and larvae to explore the machinery that targets Armadillo for destruction. Using RNAi in Drosophila S2 cells to examine which SCF components are essential for Armadillo destruction, we find that Roc1/Roc1a is essential for regulating Armadillo stability, and that in these cells the only F-box protein playing a detectable role is Slimb. Second, we find that while embryonic and larval Drosophila tissues use the same destruction complex proteins, the response of these tissues to destruction complex inactivation differs, with Armadillo levels more elevated in embryos. We provide evidence consistent with the possibility that this is due to differences in armadillo mRNA levels. Third, we find that there is no correlation between the ability of different APC2 mutant proteins to negatively regulate Armadillo levels, and their recently described function in positively-regulating Wnt signaling. Finally, we demonstrate that APC proteins lacking the N-terminal Armadillo-repeat domain cannot restore Armadillo destruction but retain residual function in negatively-regulating Wnt signaling.Conclusions/SignificanceWe use these data to refine our model for how Wnt signaling is regulated during normal development.

Homo-oligomerization Is Essential for Toll/Interleukin-1 Receptor Domain-containing Adaptor Molecule-1-mediated NF-κB and Interferon Regulatory Factor-3 Activation

Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM-1, also named TIR domain-containing adaptor-inducing interferon (IFN)-beta or TRIF)) is a signaling adaptor of Toll-like receptor (TLR) 3/4 that activates the transcription factors, interferon regulatory factor-3 (IRF-3) and NF-kappaB leading to inducing IFN-beta production. The mechanisms by which TICAM-1 is activated by TLR3/4 to serve as a signaling platform are unknown. In this study, we show that homo-oligomerization of TICAM-1 is critical for TICAM-1-mediated activation of NF-kappaB and IRF-3. Both TIR and C-terminal domain of TICAM-1 mediated TICAM-1 oligomerization. Pro(434) located in the TIR domain and the C-terminal region, with the exception of the RIP homotypic-interacting motif, were determinants of TICAM-1 oligomerization. Mutation of TIR domain (P434H) or deletion of C-terminal domain greatly reduced TICAM-1-mediated NF-kappaB and IFN-beta promoter activation. TICAM-1 oligomerization at either the TIR domain or the C-terminal region resulted in recruitment of tumor necrosis factor receptor-associated factor 3, a downstream signaling molecule essential for TICAM-1-mediated IRF-3 activation, but not recruitment of the IRF-3 kinase complex, NF-kappaB-activating kinase-associated protein 1 and TANK-binding kinase 1. In addition, RIP homotypic-interacting motif mutant, which possesses two oligomerization motifs but not the RIP1 binding motif, also failed to recruit NF-kappaB-activating kinase-associated protein 1 and TANK-binding kinase 1. Thus, full activation and formation of TICAM-1 signalosomes requires oligomerization induced at two different sites and RIP1 binding.

SnapShot: Wnt/β-Catenin Signaling

Molecules labeled in green and blue play positive and negative roles in Wnt/β-catenin signaling, respectively. Molecules that are labeled in both colors have dual roles.(Top left) Wnt biogenesis. A lipid modification is added to Wnt ligands by Porc in the endoplasmic reticulum (ER). Wnt ligands are glycosylated in the ER and Golgi and require Wls (also known as Evi) to traffic through the Golgi to the plasma membrane. The retromer complex is also important for Wnt secretion, particularly for long-range signaling. Mature Wnt ligands may interact with lipoprotein particles. Proteoglycans Dally and Kny/Glypican may also facilitate Wnt distribution.(Bottom right) Wnt receptor biogenesis. In cells that respond to Wnt the ER protein MESD is required for folding and trafficking of the Wnt receptor LRP5/6 to the plasma membrane, and the ER protein Shisa prevents folding and trafficking of the Fz protein to the plasma membrane.(Right) Wnt/β-catenin signaling OFF. sFRP and WIF1 directly bind Wnt ligands and prevent Wnts from binding to receptors. SOST/WISE, CTGF/Cyr61 bind to LRP6 (CTGF may also bind to Fz) to prevent the formation of the Wnt-Fz-LRP5/6 receptor complex. DKK binds to and inhibits LRP5/6 in cooperation with the KRM receptor. In the absence of Wnt signaling, the scaffolding protein Axin and tumor suppressor APC form a β-catenin destruction complex that binds cytosolic β-catenin and facilitates sequential phos-phorylation of β-catenin by CK1 (at S45) and GSK3 (at S33/S37/T41). The tumor suppressor WTX may also reside in this complex. Phosphorylated β-catenin is recognized by β-Trcp and ubiquitinated for degradation by the proteasome. In the nucleus, TCF assembles a transcriptional repressor complex to silence Wnt target genes via recruiting Gro, CtBP, and HDACs. Residual β-catenin is exported from the nucleus by RanBP3 and APC or bound by CBY or ICAT that prevents β-catenin association with TCF/LEF.(Left) Wnt/β-catenin signaling ON. The Wnt ligand binds to Fz and LRP5/6 receptors to form a Fz-LRP5/6 complex. Dally and Kny can also bind Wnt and enrich Wnt concentration locally or help Wnt gradient distribution. Rspo proteins and Norrin are secreted agonists that bind to LRP5/6 and/or Fz to activate Wnt/β-catenin signaling. Formation of the Fz-LRP6 complex via Dvl promotes LRP6 phosphorylation by GSK3 and CK1γ and other CK1. Fz binds Dvl and phosphorylated LRP6 recruits Axin to the plasma membrane, resulting in inhibition of β-catenin phosphorylation/degradation by an as yet unknown mechanism. Fz-LRP6-Dvl aggregation may be involved. LRP6 association with caveolin may promote its endocytosis and signaling. Translocation of Axin is facilitated by MACF1. Trimeric G proteins may act between Fz and Dvl, and β-arrestin may associate with Dvl and Axin. Stabilized β-catenin is translocated to the nucleus where it binds TCF/LEF and recruits coactivators such as the Lgs/Pygo complex, CBP/p300, Brahma, MED12/Mediator, and the PAF1/Hyrax complex to initiate RNA transcription and elongation. TCF/β-catenin controls the expression of many genes that affect cell proliferation (i.e.,

Repression of Wnt/β-catenin signaling in the anterior endoderm is essential for liver and pancreas development

The liver and pancreas are specified from the foregut endoderm through an interaction with the adjacent mesoderm. However, the earlier molecular mechanisms that establish the foregut precursors are largely unknown. In this study, we have identified a molecular pathway linking gastrula-stage endoderm patterning to organ specification. We show that in gastrula and early-somite stage Xenopus embryos, Wnt/beta-catenin activity must be repressed in the anterior endoderm to maintain foregut identity and to allow liver and pancreas development. By contrast, high beta-catenin activity in the posterior endoderm inhibits foregut fate while promoting intestinal development. Experimentally repressing beta-catenin activity in the posterior endoderm was sufficient to induce ectopic organ buds that express early liver and pancreas markers. beta-catenin acts in part by inhibiting expression of the homeobox gene hhex, which is one of the earliest foregut markers and is essential for liver and pancreas development. Promoter analysis indicates that beta-catenin represses hhex transcription indirectly via the homeodomain repressor Vent2. Later in development, beta-catenin activity has the opposite effect and enhances liver development. These results illustrate that turning Wnt signaling off and on in the correct temporal sequence is essential for organ formation, a finding that might directly impact efforts to differentiate liver and pancreas tissue from stem cells.

Cis-regulatory processing of Notch signaling input to the sea urchin glial cells missing gene during mesoderm specification

The glial cells missing regulatory gene of Strongylocentrotus purpuratus ( spgcm) was proposed earlier to be the genomic target of Delta/Notch (D/N) signaling required for specification of the mesodermal precursors of pigment cells. Here, we show that microinjection of a spgcm antisense morpholino oligonucleotide results in larvae without pigment cells. Microinjection of an mRNA encoding a dominant negative form of Suppressor of Hairless (dn-Su(H)) results in reduced levels of spgcm mRNA, disruption of mesodermal founder cell specification and failure to produce pigment cells. These results confirm that this gene is required for pigment cell specification. Three cis-regulatory modules of the spgcm gene were identified, which when incorporated in a GFP expression construct recapitulate the early expression pattern of this gene. Spatial expression of this GFP expression construct is severely disrupted by co-expression of dn-Su (H) mRNA, confirming that spgcm is a direct target of canonical N signaling mediated through Su(H) inputs. cis-perturbation analysis by mutation of consensus Su(H) sites identified a conserved motif paired-site and a lone site in the middle module that function both to drive expression in SMC precursors which receive the Delta signal and to repress expression in ectopic locations which lack this signal. While these Su(H) target sites provide the cis-regulatory architecture with the core of an N signaling transcriptional response switch, both the on and off outputs from this module require additional inputs.

Rescue of a mutant G-protein by substrate-assisted catalysis.

Signaling by guanine-nucleotide-binding proteins (G-proteins) occurs when they are charged with GTP, while hydrolysis of the bound nucleotide turns the signaling off. Despite a wealth of biochemical and structural information, the mechanism of GTP hydrolysis by G-proteins remains controversial. We have employed substrate-assisted catalysis as a novel approach to study catalysis by G-proteins. In these studies, we have used diaminobenzophenone-phosphonoamidate-GTP, a unique GTP analog bearing the functional groups that are missing in the GTPase-deficient [Leu227]G(s alpha) mutant. This mutant, found in various human tumors, fails to hydrolyze GTP for an extended period. In contrast, the GTP analog is hydrolyzed by this mutant and by the wild-type enzyme at the same rate. On the other hand, modification of G(s alpha) by cholera toxin, which catalyses ADP-ribosylation of Arg201 of G(s alpha), decreased the rates of hydrolysis of both GTP and its analog by 95%. These results attest to the specificity of the GTP analog as a unique substrate for the [Leu227]G(s alpha) mutant and to the essential role of Gln227 in GTP hydrolysis. Furthermore, the finding that the GTP analog was hydrolyzed at the same rate as GTP by the wild-type enzyme, favors a model in which formation of a pentavalent transition state intermediate, presumably stabilized by the catalytic glutamine, is not the rate-limiting step of the GTPase reaction.