A Drosophila In Vivo Screen Identifies Store-Operated Calcium Entry as a Key Regulator of Adiposity

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An In Vivo C. elegans Model System for Screening EGFR-Inhibiting Anti-Cancer Drugs

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.     

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.     

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis

BackgroundThe C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis.ResultsWe found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1.ConclusionsPTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.     

Growth control by EGF repeats of the C. elegans Fibulin-1C isoform

Fibulin is a broadly conserved component of the extracellular matrix (ECM). Previous studies have shown that Caenorhabditis elegans FIBULIN-1 (FBL-1) controls the width of the gonad (Hesselson, D., C. Newman, K.W. Kim, and J. Kimble. 2004. Curr. Biol. 14:2005-2010; Kubota, Y., R. Kuroki, and K. Nishiwaki. 2004. Curr. Biol. 14:2011-2018; Muriel, J.M., C. Dong, H. Hutter, and B.E. Vogel. 2005. Development. 132: 4223-4234). In this study, we report that FBL-1 also controls developmental growth and that one isoform of fibulin-1, called FBL-1C, controls both functions by distinct mechanisms. A large FBL-1C fragment, including both epidermal growth factor (EGF) and fibulin-type C domains, is responsible for constraining gonadal width, but a much smaller fragment containing only two complete EGF repeats (EGF1-2C+) is critical for developmental growth. We suggest that the larger fragment serves a scaffolding function to stabilize the basement membrane and that the smaller fragment provides a regulatory function at the cell surface or within the ECM to control growth.     

Yeast Three-Hybrid Screen Identifies TgBRADIN/GRA24 as a Negative Regulator of Toxoplasma gondii Bradyzoite Differentiation

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite’s life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen.     

The C. elegans ezrin-radixin-moesin protein ERM-1 is necessary for apical junction remodelling and tubulogenesis in the intestine

Members of the ezrin-radixin-moesin (ERM) family of proteins have been found to serve as linkers between membrane proteins and the F-actin cytoskeleton in many organisms. We used RNA interference (RNAi) approach to assay ERM proteins of the Caenorhabditis elegans genome for a possible involvement in apical junction (AJ) assembly or positioning. We identify erm-1 as the only ERM protein required for development and show, by multiple RNA interference, that additional four-point one, ezrin-radixin-moesin (FERM) domain-containing proteins cannot compensate for the depletion of ERM-1. ERM-1 is expressed in most if not all cells of the embryo at low levels but is upregulated in epithelia, like the intestine. ERM-1 protein co-localizes with F-actin and the intermediate filament protein IFB-2 at the apical cell cortex. ERM-1 depletion results in intestine-specific phenotypes like lumenal constrictions or even obstructions. This phenotype arises after epithelial polarization of intestinal cells and can be monitored using markers of the apical junction. We show that the initial steps of epithelial polarization in the intestine are not affected in erm-1(RNAi) embryos but the positioning of apical junction proteins to an apico-lateral position arrests prematurely or fails, resulting in multiple obstructions of the intestinal flow after hatching. Mechanistically, this phenotype might be due to an altered apical cytoskeleton because the apical enrichment of F-actin filaments is lost specifically in the intestine. ERM-1 is the first protein of the apical membrane domain affecting junction remodelling in C. elegans. ERM-1 interacts genetically with the catenin-cadherin system but not with the DLG-1 (Discs large)-dependent establishment of the apical junction.     

A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.     

The M33 Chemokine Receptor Homolog of Murine Cytomegalovirus Exhibits a Differential Tissue-Specific Role during In Vivo Replication and Latency

M33, encoded by murine cytomegalovirus (MCMV), is a member of the UL33 homolog G-protein-coupled receptor (GPCR) family and is conserved across all the betaherpesviruses. Infection of mice with recombinant viruses lacking M33 or containing specific signaling domain mutations in M33 results in significantly diminished MCMV infection of the salivary glands. To determine the role of M33 in viral dissemination and/or infection in other tissues, viral infection with wild-type K181 virus and an M33 mutant virus, ΔM33B_T2, was characterized using two different routes of inoculation. Following both intraperitoneal (i.p.) and intranasal (i.n.) inoculation, M33 was attenuated for infection of the spleen and pancreas as early as 7 days after infection. Following i.p. inoculation, ΔM33B_T2 exhibited a severe defect in latency as measured by a diminished capacity to reactivate from spleens and lungs in reactivation assays (P < 0.001). Subsequent PCR analysis revealed markedly reduced ΔM33B_T2 viral DNA levels in the latently infected spleens, lungs, and bone marrow. Following i.n. inoculation, latent ΔM33B_T2 viral DNA was significantly reduced in the spleen and, in agreement with results from i.p. inoculation, did not reactivate from the spleen (P < 0.001). Furthermore, in vivo complementation of ΔM33B_T2 virus replication and/or dissemination to the salivary glands and pancreas was achieved by coinfection with wild-type virus. Overall, our data suggest a critical tissue-specific role for M33 during infection in the salivary glands, spleen, and pancreas but not the lungs. Our data suggest that M33 contributes to the efficient establishment or maintenance of long-term latent MCMV infection.Since the discovery of the G protein-coupled receptors (GPCRs) encoded by the betaherpesviruses, there has been intense speculation on the biological role these viral proteins play during infection (15, 16, 22). Human cytomegalovirus (HCMV), a betaherpesvirus, is a ubiquitous pathogen that asymptomatically infects humans and establishes a long-term persistent infection. HCMV is life-threatening, however, to immunocompromised individuals, such as neonates, AIDS patients, and transplant recipients. HCMV, similar to a number of herpesviruses, encodes viral genes that are predicted to impact virus-host interactions that may promote efficient long-term infection of the host. The CMVs encode genes for proteins that potentially enhance viral dissemination and replication and promote immune evasion by mimicry of host functions that influence the conditions of primary infection, the virus-specific immune response, and even long-term host control of persistent or latent infection (reviewed in references 1, 44, and 68).HCMV encodes four GPCRs (UL33, UL78, US28, and US27) which share homology to host chemokine receptors (16). This suggests that these virally encoded chemokine receptors may function similarly to their cellular receptor counterparts. Chemokines are chemoattractant cytokines that bind and activate chemokine receptors that are on the surfaces of cells. Host chemokine receptors then mediate the activation of cellular signaling pathways and cell migration to sites of inflammation by transmitting signals through G proteins (56, 70). In humans, approximately 50 chemokines and 20 chemokine receptors have been identified, many of which have close homologs in mice and other species (39). Chemokines are divided into two classes, lymphoid chemokines, which are constitutively expressed and involved in lymphoid tissue organization, and inflammatory chemokines, which are induced following infection and part of the inflammatory response (21, 39, 51). Growing evidence indicates that chemokines play a critical role in the host response to infection and inflammation during both the innate and adaptive immune responses (26), thus suggesting that the betaherpesviruses have “hijacked” the chemokine receptors from the host genome to subvert or alter these responses during infection. Besides chemokine receptors, HCMV also encodes a CXC chemokine (UL146) that induces the migration of neutrophils (48); a second CXC chemokine homolog (UL147) whose function is not yet known; a viral CC chemokine (UL131) that is critical for infection of macrophages, endothelial cells, and epithelial cells (25, 57, 73); and a RANTES decoy protein (72). A CC chemokine (vMCK or m131/129) is also encoded by murine CMV (MCMV), and a homolog in rat CMV ([RCMV] r131) that promotes monocyte-associated viremia (20, 37, 59, 60). The MCMV m131/129 chemokine was shown to recruit myelomonocytic progenitors from the bone marrow, perhaps to facilitate cell-type-specific viremia (46). Clearly, the CMVs have invested a great deal of effort into manipulating or subverting the host chemokine system, thus making it reasonable to speculate that these viral members of the chemokine system play an important role during CMV pathogenesis.Of the HCMV-encoded GPCRs, US28 has been well characterized in vitro and functions as a bona fide chemokine receptor, whereas much less is known about the receptor activity of US27, UL33, and UL78. US28 binds and sequesters CC chemokines, induces smooth-muscle cell migration, and constitutively activates signaling pathways (5, 7-9, 42, 52, 64, 67, 71). US28 and US27 are found only in primate CMVs, whereas both UL33 and UL78 are highly conserved across all betaherpesvirus genomes, suggesting an important evolutionary function for UL33 and UL78 during CMV infection. Two other betaherpesviruses, human herpesviruses 6 and 7 (HHV6 and HHV7), encode homologs to the UL33 and UL78 receptors, U12 and U51, respectively. The U12 receptors of HHV6 and HHV7 (34, 45, 66) and the HHV6-encoded U51 receptor (22) exhibit chemokine binding activity. UL33, along with its rodent CMV homologs, M33 (MCMV) and R33 (RCMV), constitutively activates signaling pathways (13, 23, 71). M33 induces smooth-muscle cell migration (39), similar to US28-mediated smooth-muscle cell migration (64). Thus, members of the UL33 family potentially function during viral infection by modulating or influencing the composition of leukocytes at sites of infection, the migration of infected cells or infiltrating leukocytes, or modulation of intracellular signaling pathways.Due to the species specificity of CMV, the in vivo role of the HCMV-encoded GPCRs cannot be addressed. However, the importance of UL33 and UL78 for viral dissemination and virulence in vivo has been indicated by disruption of the viral homologs in MCMV and RCMV (6, 19, 36, 47). Disruption of the UL33, M33, and R33 genes demonstrated that they are dispensable for replication in vitro, indicating that the UL33 family members are not required for replication or cell entry in at least some cell types (6, 19, 40). Infection of mice with M33-deficient MCMV or infection of rats with R33-deficient RCMV results in highly attenuated viruses and diminished infection of the salivary glands. The RCMV R33 protein also appears to play a role in virulence since rats infected with an R33 deletion virus had a lower mortality rate (6). More recently, constitutive M33-mediated activation of signaling pathways was shown to be essential for MCMV infection of salivary glands (14). Significantly, the UL33 protein partially rescued the defect in salivary gland infection attributed to disruption of M33, indicating the evolutionary conservation of function between the HCMV (UL33) and MCMV (M33) chemokine receptor homologs.In this paper, the role of M33 is further investigated using two routes of infection to assess viral dissemination and viral replication kinetics at different tissue sites, the numbers of infected cells following infection, and the possibility that M33 plays a role during latent infection. In addition to the critical role that M33 plays in salivary gland infection, this study reveals that M33 is important for MCMV infection of the spleen and the pancreas but not the lungs. Significantly, our studies provide preliminary evidence that disruption of M33 leads to reduced latent viral load in the spleen, lungs, and bone marrow, perhaps due to defects in the establishment and/or maintenance of latent infection. Lastly, we demonstrate that the tissue defects observed during acute infection with an M33 mutant virus (ΔM33B_T2) can be complemented in vivo when mice are coinfected with ΔM33B_T2 virus and wild-type MCMV. Taken together, our findings indicate that M33 plays a critical tissue-specific role during acute MCMV infection and, importantly, contributes to the efficient establishment or maintenance of latent MCMV infection.     

Transient Receptor Potential Ankyrin 1 Receptor Activation In Vitro and In Vivo by Pro-tussive Agents: GRC 17536 as a Promising Anti-Tussive Therapeutic

Cough is a protective reflex action that helps clear the respiratory tract which is continuously exposed to airborne environmental irritants. However, chronic cough presents itself as a disease in its own right and despite its global occurrence; the molecular mechanisms responsible for cough are not completely understood. Transient receptor potential ankyrin1 (TRPA1) is robustly expressed in the neuronal as well as non-neuronal cells of the respiratory tract and is a sensor of a wide range of environmental irritants. It is fast getting acceptance as a key biological sensor of a variety of pro-tussive agents often implicated in miscellaneous chronic cough conditions. In the present study, we demonstrate in vitro direct functional activation of TRPA1 receptor by citric acid which is routinely used to evoke cough in preclinical and clinical studies. We also show for the first time that a potent and selective TRPA1 antagonist GRC 17536 inhibits citric acid induced cellular Ca⁺² influx in TRPA1 expressing cells and the citric acid induced cough response in guinea pigs. Hence our data provides a mechanistic link between TRPA1 receptor activation in vitro and cough response induced in vivo by citric acid. Furthermore, we also show evidence for TRPA1 activation in vitro by the TLR4, TLR7 and TLR8 ligands which are implicated in bacterial/respiratory virus pathogenesis often resulting in chronic cough. In conclusion, this study highlights the potential utility of TRPA1 antagonist such as GRC 17536 in the treatment of miscellaneous chronic cough conditions arising due to diverse causes but commonly driven via TRPA1.     

A Loss-of-Function Screen for Phosphatases that Regulate Neurite Outgrowth Identifies PTPN12 as a Negative Regulator of TrkB Tyrosine Phosphorylation

Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. “Classical” protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence the relevance of considering all phosphatase families when mining for potentially druggable targets.