org Interactive Fly, Drosophila

Toll


REGULATION

Protein Interactions

Interactions with Spätzle

spätzle is a maternal effect gene required in the signal transduction pathway that establishes the dorsal-ventral pattern of the Drosophila embryo. spätzle acts immediately upstream of the membrane protein Toll in the genetic pathway, suggesting that spätzle could encode the ventrally localized ligand that activates the receptor activity of Toll. Proteolytic processing of the Spätzle protein is confined to the ventral side of the embryo; the localization of processed Spätzle determines where the receptor Toll will be active (Morisato, 1994 and Roth, 1994).

By transplantation of perivitelline fluid the presence of three separate activities present in the perivitelline fluid can be shown to restore dorsal-ventral polarity to mutant easter, snake, and spätzle embryos, respectively. These activities are not capable of defining the polarity of the dorsal-ventral axis; instead they restore structures according to the intrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventral formation of a ligand for the Toll protein. This process requires serine proteolytic activity; the injection of serine protease inhibitors into the perivitelline space of wild-type embryos results in the formation of dorsalized embryos (Stein, 1992).

A soluble, extracellular factor found to induce ventral structures at the site where it is injected into the extracellular space of the early Drosophila embryo has the properties predicted for a ligand for Toll. Using a bioassay to follow activity, a 24 x 10(3) M(r) protein has been purified. The purified protein is recognized by antibodies to the C-terminal half of the Spätzle protein. The purified protein is smaller than the primary translation product of spätzle, suggesting that proteolytic processing of Spätzle is required to generate the localized, active form of the protein (Schneider, 1994).

Microbial infection activates two distinct intracellular signaling cascades in the immune-responsive fat body of Drosophila. Gram-positive bacteria and fungi predominantly induce the Toll signaling pathway, whereas Gram-negative bacteria activate the Imd pathway. Loss-of-function mutants in either pathway reduce the resistance to corresponding infections. Genetic screens have identified a range of genes involved in these intracellular signaling cascades, but how they are activated by microbial infection is largely unknown. Activation of the transmembrane receptor Toll requires a proteolytically cleaved form of an extracellular cytokine-like polypeptide, Spatzle, suggesting that Toll does not itself function as a bona fide recognition receptor of microbial patterns. This is in apparent contrast with the mammalian Toll-like receptors and raises the question of which host molecules actually recognize microbial patterns to activate Toll through Spatzle. A mutation is described in this study that blocks Toll activation by Gram-positive bacteria and significantly decreases resistance to this type of infection. The mutation semmelweis (seml) inactivates the gene encoding a peptidoglycan recognition protein (PGRP-SA). Interestingly, seml does not affect Toll activation by fungal infection, indicating the existence of a distinct recognition system for fungi to activate the Toll pathway (Michel, 2001).

The sequence of the PGRP-SA complementary DNAs from seml and wild-type flies was compared. In the seml cDNA there is a transition of guanine to adenine at position 174, which results in the change of cysteine 80 into a tyrosine. This mutation affects a region of amino acids (called the PGRP domain) that is extremely conserved among the members of the PGRP family of vertebrates and invertebrates. This cysteine is conserved in more than 90% of the PGRPs, a feature shared by only 5% of the amino acids that form the PGRP domain. Furthermore, a cysteine is present in close proximity (Cys 74) that might engage in a disulphide bridge with Cys 80. The seml mutation would disrupt such a bridge and affect the three-dimensional structure of the protein. To ascertain that the mutation of Cys 80 to Tyr 80 is responsible for the seml phenotypes, rescue experiments were undertaken by overexpressing wild-type dPGRP-SA cDNA, using the ubiquitous driver DaughterlessGal4 (DaGal4). Adult seml;DaGal4-UAS PGRP-SA flies have a restored ability to induce drosomycin after M. luteus infection and are no longer susceptible to Gram-positive infection. In contrast, seml;DaGal4 flies show phenotypes identical to seml flies. These results unambiguously demonstrate that the inability of seml mutant flies to resist Gram-positive infection results from the inactivation of the PGRP-SA gene (Michel, 2001).

The deduced sequence of the PGRP-SA gene indicates the presence of a putative signal peptide, suggesting that the PGRP-SA protein is a secreted protein present in the hemolymph or possibly associated with the extracellular matrix. It was reasoned that if the PGRP-SA protein is present in the hemolymph, it should be possible to rescue the seml phenotype by transferring wild-type hemolymph into the mutant flies. Indeed, when wild-type hemolymph was injected into seml flies, the recipient flies became capable of expressing drosomycin after challenge by M. luteus. These results demonstrate that PGRP-SA is a protein circulating in the hemolymph, where the molecular recognition with the Gram-positive bacterial cell wall components can occur (Michel, 2001).

The data demonstrate that activation of the Toll pathway by Gram-positive bacterial infection is mediated by a circulating peptidoglycan recognition protein. This is the first demonstration of an in vivo function for a pattern-recognition receptor in invertebrate innate immunity. In view of the relatively large number of genes (12) encoding peptidoglycan recognition proteins in Drosophila, it was surprising to see that the mutation of one of these genes was sufficient to abolish Toll activation by Gram-positive bacteria. No other circulating protein can substitute for PGRP-SA in the case of M. luteus and the three others strains tested. How the recognition of M. luteus by PGRP-SA leads to the activation of Toll by cleaving Spatzle remains to be determined in molecular terms. PGRP-SA has no protease domain and it is speculated that conformational changes, induced on binding to microbial patterns, may activate associated proteases, analogous to that described for mannose-binding lectin associated proteases (MASPs) in the activation of the lectin pathway. Finally, the observation that seml mutant flies remain capable of mounting a Toll-dependent antifungal response clearly shows that the upstream mechanisms leading to activation of Toll are specific for each class of microorganisms. The versatility of the Toll-like receptors on mammalian immune-responsive cells is probably paralleled by a versatility of circulating pattern-recognition receptors in Drosophila (Michel, 2001).

The Toll family of receptors is required for innate immune response to pathogen-associated molecules, but the mechanism of signaling is not entirely clear. In Drosophila the prototypic Toll regulates both embryonic development and adult immune response. The host protein Spatzle can function as a ligand for Toll because Spatzle forms a complex with Toll in transgenic fly extracts and stimulates the expression of a Toll-dependent immunity gene, drosomycin, in adult flies. Constitutively active mutants of Toll form multimers that contain intermolecular disulfide linkages. These disulfide linkages are critical for the activity of one of these mutant receptors, indicating that multimerization is essential for the constitutive activity. Furthermore, systematic mutational analysis revealed that a conserved cysteine-containing motif, different from the cysteines used for the intermolecular disulfide linkages, serves as a self-inhibitory module of Toll. Deleting or mutating this cysteine-containing motif leads to constitutive activity. This motif is located just outside the transmembrane domain and may provide a structural hindrance for multimerization and activation of Toll. Together, these results suggest that multimerization may be a regulated, essential step for Toll-receptor activation (Hu, 2004)

Activation of Drosophila Toll during fungal infection by a blood serine protease

Drosophila host defense to fungal and Gram-positive bacterial infection is mediated by the Spaetzle/Toll/cactus gene cassette. It has been proposed that Toll does not function as a pattern recognition receptor per se but is activated through a cleaved form of the cytokine Spaetzle. The upstream events linking infection to the cleavage of Spaetzle have long remained elusive. This study reports the identification of a central component of the fungal activation of Toll. Ethylmethane sulfonate-induced mutations in the persephone gene, which encodes a previously unknown serine protease, block induction of the Toll pathway by fungi and resistance to this type of infection (Ligoxygakis, 2002).

The Drosophila host defense is a multifaceted process, which involves the challenge-dependent synthesis of potent antimicrobial peptides by the fat body, a functional equivalent of the mammalian liver. Two intracellular signaling pathways mediate the synthesis of these peptides: the Toll and the Imd pathway. Toll is activated during fungal and Gram-positive bacterial defenses, and the Imd pathway is predominantly activated by Gram-negative bacterial infections. Toll was initially identified as a gene that controls the establishment of dorsoventral polarity in early embryogenesis. A proteolytically cleaved form of the cysteine-knot growth factor Spaetzle (Spz) functions as the extracellular ligand of Toll in both embryonic development and the immune response. During embryonic patterning, Spz cleavage is achieved by the sequential activation of three serine proteases required in the germ line: Gastrulation defective, Snake, and Easter. However, in null mutants of these proteases, challenged-induced activation of the Toll pathway is not affected, as illustrated by wild-type induction of the antifungal peptide drosomycin. The implication is that infection may activate some other protease(s) that cleaves Spz to its active form. Immune-induced cleavage of Spz by blood serine proteases is conceptually similar to blood coagulation or complement activation, where inappropriate activation is prevented by the action of serine protease inhibitors (serpins). Flies mutant for the blood serpin Necrotic (Nec) exhibit a constitutively activated antifungal host defense, and Spz is constitutively cleaved in this mutant background (Levashina, 1999). The nec pleiotropic phenotypes include spontaneous melanization, cellular necrosis, and death in early adulthood. These changes probably all reflect a role of the nec serpin gene in controlling activation of one or more proteolytic cascades (Ligoxygakis, 2002).

To identify components of the antifungal response cascade that activate Spz, the first chromosome was screened for ethylmethane sulfonate-induced suppressors of the nec phenotypes. From 9700 mutagenized male flies trans-heterozygous for nec, five suppressors were isolated that belong to the same complementation group. This suppressor mutation was called persephone (psh). Mutations in psh suppressed all nec phenotypes. In particular, psh;nec double mutants displayed a life-span comparable to that of wild-type (WT) flies, showed no spontaneous melanization, and did not express drosomycin in a constitutive manner. When challenged with fungi, psh mutants exhibited a severely reduced level of drosomycin transcription as compared with WT flies. Induction of drosomycin by Gram-positive bacteria was at WT levels in psh mutants. Finally, expression of diptericin, which is controlled by the Imd pathway, was not affected following Gram-negative bacterial infection (Ligoxygakis, 2002).

It was further noted that psh flies were highly susceptible to fungal infections, behaving in this respect as Toll pathway mutants. Conversely, psh flies showed a WT pattern of survival after immune challenge by Gram-positive bacteria. As expected, psh flies were resistant to Gram-negative bacterial infection, which activates the Imd pathway (Ligoxygakis, 2002).

For epistasis studies, fly lines were used overexpressing a cleaved form of Spz through the UAS/GAL4 system. These flies exhibit a challenge-independent expression of drosomycin. The levels of drosomycin transcription after overexpression of spz were notably similar in psh mutants and in WT flies. This result indicates that the psh mutation inactivates a gene upstream of spz (Ligoxygakis, 2002).

With a deficiency kit spanning the first chromosome, the psh mutation was mapped to the chromosomal region 17A-17B. This region contains two serine protease genes, CG6361 and CG6367. The genomic sequence of the CG6367 protease in psh5 was compared with that of WT flies and noted the transition of a G nucleotide to an A in the sequence encoding the conserved 'serine signature' sequence (Gly-Asp-Ser-Gly-Gly-Pro), which results in a Gly to Glu change at position 340. In the sequence of psh1 and psh4, a transition of a C to a T was observed, which is expected to result in the change of the His of the catalytic triad to a Tyr at position 187 (Ligoxygakis, 2002).

To ensure that these mutations were responsible for the observed phenotypes in psh mutants, rescue experiments were undertaken using the UAS/GAL4 system with the female fat body-specific yolkGAL4 as a driver. Itis noted that UAS-CG6367/yolkGAL4 flies constitutively express drosomycin. Furthermore, it was observed that overexpression of the CG6367 protease restores the ability of psh flies to respond to fungal infection. Neither an activated form of Easter nor the CG6361 protease contained in the deficiency that uncovers the psh locus were able to rescue the observed sensitivity to fungi. These results indicate that CG6367 is the serine protease responsible for the psh phenotype (Ligoxygakis, 2002).

The deduced sequence of the PSH protein indicates the presence of a putative signal peptide (amino acids 1 to 20), suggesting that the protein is secreted and present in the hemolymph, as has been shown for Nec. Transfer of hemolymph from nec flies to WT flies carrying a drosomycin-GFP (drs-GFP) reporter resulted in expression of drs-GFP. In contrast, transfer of hemolymph from psh;nec flies did not induce drosomycin expression. This indicates that the blood-borne factor responsible for Toll activation observed in nec hemolymph transfer is suppressed in psh;nec flies. Finally, transfer of hemolymph from flies overexpressing WT psh (UAS-CG6367/daughterlessGAL4) to drs-GFP flies induced challenge-independent expression of drosomycin. These results confirm the presence of immune-responsive components of the system in the hemolymph (Ligoxygakis, 2002).

The blood serine protease Persephone is the first identified component of the cascade, which was hypothesized to activate Toll following an immune challenge. Serine proteases are initially synthesized as inactive zymogens containing an NH2-terminal prodomain and a COOH-terminal catalytic domain. Activation requires proteolytic cleavage of the zymogen at a defined site by a specific activating protease or a nonenzymatic ligand. The sequence of PSH also predicts an NH2-terminal prodomain. This prodomain contains a CLIP module most homologous to those in Easter, Snake, the horseshoe crab proclotting factor, and the Bombyx mori prophenoloxidase-activating enzyme. Thus, common organizing principles may direct hemolymph clotting, immune response, and developmental serine protease cascades in arthropods. psh is the first described mutation to specifically impair Toll-dependent induction of drosomycin by fungal infection in Drosophila without affecting Gram-positive bacterial induced responses. Mutations have been reported recently that affect activation of the Toll pathway by Gram-positive bacteria and activation of the Imd pathway by Gram-negative bacteria. The mutated genes encode members of the family of soluble or membrane proteins referred to as peptidoglycan recognition proteins (PGRPs), in reference to their initial discovery as Gram-positive interacting proteins. Toll activation by Gram-positive bacteria is mediated by a soluble PGRP, whereas that of Imd by Gram-negative infection involves a putative membrane PGRP. Taken together, the results on the psh mutation and those on mutations in the soluble PGRP-SA and the putative membrane PGRP-LC now define three distinct upstream pathways mediating response to fungal infections and to infections by Gram-positive or Gram-negative bacteria. These data set the stage for a detailed analysis of the events leading from recognition of infection to activation of intracellular signaling pathways and consequent transcription of appropriate groups of genes concurring to fight the respective infections. Whereas PGRPs can be considered as bona fide pattern recognition receptors, psh has no microbial pattern recognition-binding domain. It is anticipated that an as-yet unidentified, upstream fungal pattern recognition receptor functions to activate the protease function of psh (Ligoxygakis, 2002).

Drosophila immunity: A large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation

Unlike mammalian Toll-like receptors, the Drosophila Toll receptor does not interact directly with microbial determinants but is rather activated upon binding a cleaved form of the cytokine-like molecule Spatzle (Spz). During the immune response, Spz is thought to be processed by secreted serine proteases (SPs) present in the hemolymph that are activated by the recognition of gram-positive bacteria or fungi. In the present study, an in vivo RNAi strategy was used to inactivate 75 distinct Drosophila SP genes. This collection was then screened for SPs regulating the activation of the Toll pathway by gram-positive bacteria. This study reports the identification of five novel SPs that function in an extracellular pathway linking the recognition proteins GNBP1 and PGRP-SA to Spz. Interestingly, four of these genes are also required for Toll activation by fungi, while one is specifically associated with signaling in response to gram-positive bacterial infections. These results demonstrate the existence of a common cascade of SPs upstream of Spz, integrating signals sent by various secreted recognition molecules via more specialized SPs (Kambris, 2006).

Until recently, only one SP, Psh, has been shown to act upstream of Toll in response to fungi. Via a large-scale RNAi screen, five novel SPs have been identified that regulate Toll activity in response to gram-positive bacterial infections. Three of them, Spirit (for Serine Protease Immune Response Integrator), Grass, and SPE, are functional chymotrypsin-like SPs containing a Clip domain N-terminal to the catalytic domain. The Clip domain is exclusively found in insect SPs and is believed to play a regulatory role in the sequential activation of SPs. For instance, it is present in Psh as well as in Snake and Easter, two SPs that participate in the processing of Spz during embryonic development. In contrast to spirit and SPE (Spatzle Processing Enzyme), grass (Gram-positive Specific Serine protease) is required to resist gram-positive bacterial but not fungal infection, and its knock-down induces a phenotype similar to those induced by GNBP1 or PGRP-SA mutations. This is in agreement with a model in which Grass is activated by GNBP1 and PGRP-SA after recognition of gram-positive peptidoglycan. Grass then transmits a signal that is integrated by a common core of downstream SPs including Spirit and SPE. In agreement with a position of Grass upstream in the Toll-activating cascade, the expression of an activated form of SPE and Spirit but not Grass can trigger the antimicrobial peptide gene Drosomycin (Drs) in S2 cells. Further signal amplification could occur since both spirit and SPE are induced during the immune response in a Toll pathway-dependent manner. Thus, this combination of controlled proteolytic SPs activation and transcriptional positive feedback would ensure an adequate response to infection (Kambris, 2006).

Surprisingly, two SPs identified in in the screen, Spheroide and Sphinx1/2, are unlikely to possess proteolytic activity, because their protease-like domain lacks the catalytic serine residue. This class of SP homologs (SPHs) represents one quarter of Drosophila SP-related proteins and is thought to have regulatory functions. Several SPHs have been shown to be involved in the proteolytic cascade that regulates the cleavage of prophenoloxidase in other insects, supporting a role of this class of SP-related proteins during the immune response. Since knock-downs of spheroide and sphinx1/2 induces the same phenotype as that of SPE and spirit, it is suggested that these two SPHs may act as adaptors or regulators of SPE and Spirit, possibly by localizing the two SPs in close proximity to Spz and/or the fat-body cell membrane to promote robust activation of the Toll receptor (Kambris, 2006).

RNAi of the five SPs did not affect activation of melanization nor did it suppress lethality induced by the inactivation of spn27A. In agreement with two recent studies, RNAi of SP7 (CG3066) reduces melanization at the wound site, confirming the implication of this SP in the prophenoloxidase cascade. The RNAi of this SP did not affect Toll activation by gram-positive bacteria. Collectively, these data indicate that distinct SPs mediate melanization and Toll activation, underlining the complexity of SP cascades regulating the Drosophila immune response. Further studies, including the generation of null mutations, are required to analyze in more detail the function of these five SPs upstream of Toll (Kambris, 2006).

This study represents the first extensive in vivo RNAi screen of a large gene family in Drosophila. A key advantage of this strategy for functional genomic studies is that genes required during development can be inactivated in a tissue- and temporal-specific manner. It is especially suitable for the analysis of genes encoding secreted proteins such as SPs that function in a non-cell-autonomous manner and therefore require in vivo studies. The SP-RNAi fly collection described in this study and made available to the scientific community should pave the way for additional biochemical studies and genetic screens to further decipher the signaling pathways acting upstream of Toll as well as to identify additional important SP functions (Kambris, 2006).

Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors

The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the pattern recognition receptors PGRP-SA and GNBP1 that cooperate to detect the presence of infections in the host. This study reports that GNBP3 is a pattern recognition receptor that is required for the detection of fungal cell wall components. Strikingly, there is a second, parallel pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host (Gottar, 2006).

Fungi represent a threat to insects in the wild, with more than 700 described entomopathogenic species. Insects must have evolved responses to handle these infections. This study used Drosophila to decipher the mechanisms that stimulate immune responses to fungal infections (Gottar, 2006).

The Drosophila host response includes both cellular and humoral arms. The analysis of the humoral immune response within the framework of a septic injury model has led to the current paradigm in which two distinct intracellular transduction pathways, immune deficiency (IMD) and Toll, regulate the transcription of hundreds of genes by controlling the nuclear uptake of the NF-κB transcription factors Relish and Dorsal-related immunity factor (DIF), respectively. The classical effector molecules of the systemic humoral response, the antimicrobial peptides, are synthesized in the fat body, a functional analog of the mammalian liver, and are released into the hemolymph, where they kill invading microorganisms. One of these peptides, Drosomycin, exhibits fungicidal activities at micromolar concentrations and is active mainly on filamentous fungi. Others, such as Cecropins, Attacins, Drosocin, and Diptericin, are active mostly on Gram-negative bacteria, whereas Defensin is effective against Gram-positive bacteria (Gottar, 2006).

The IMD pathway is required for the host response against Gram-negative bacteria. Mutants in this pathway fail to express antibacterial peptides and are highly sensitive to such infections yet resist fungal and Gram-positive bacterial infections as well as wild-type flies (Gottar, 2006).

Toll is the receptor of the second intracellular transduction pathway and is activated by the binding of a proteolytically cleaved form of the Spätzle (SPZ) cytokine. Toll pathway mutants are susceptible to infections by the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae or by the opportunistic pathogen Aspergillus fumigatus. Toll is also required to resist some Gram-positive bacterial infections (Gottar, 2006).

The Drosophila immune response is adapted to the nature of the invading microorganism. The Toll pathway is induced by fungi and Gram-positive bacteria, whereas the IMD pathway is predominantly triggered upon Gram-negative bacterial challenges. These observations imply that several receptors mediate the discrimination between various types of microbial infections. Indeed, members of the peptidoglycan recognition protein (PGRP) family have been shown to be required for these distinct events. PGRP-LC, a receptor of the IMD pathway, can be activated by meso-diaminopimelic acid peptidoglycan (PGN), a compound characteristic of the cell wall of Gram-negative bacteria and of Gram-positive bacilli. PGRP-LE, a secreted member of the PGRP family, is also involved in sensing Gram-negative bacteria. In contrast, the circulating PGRP-SA receptor activates the Toll pathway upon detection of lysine-type PGN, which is a major component of the cell wall of many Gram-positive bacterial strains. The Gram-negative binding protein 1 (GNBP1) associates with PGRP-SA, and this complex is both necessary and sufficient to activate the Toll pathway upon Gram-positive challenge. The circulating PGRP-SA/GNBP1 complex activates a downstream proteolytic cascade that leads to the cleavage of the Spätzle cytokine, which then activates the Toll transmembrane receptor. Thus, PGRP-SA and GNBP1 define a Gram-positive-specific branch of Toll receptor activation. PGRP-SD also belongs to this branch and is required for the detection of other Gram-positive bacterial strains (Gottar, 2006).

This study addresses the existence of a second branch devoted to the detection of fungal infections, which also activates Toll. Indeed, mutants for the persephone (psh) gene, which encodes a clip-prodomain-containing protease, are characterized by an increased sensitivity to natural infections with the entomopathogenic fungus B. bassiana, whereas they are resistant to bacterial infections. The psh mutations had been originally isolated as suppressors of the necrotic (nec) phenotype. nec encodes a serine protease inhibitor of the serpin family, the absence of which leads to the constitutive, psh- dependent, activation of the Toll pathway. Thus, psh and nec define a fungal-specific branch of Toll receptor activation. By analogy to the Gram-positive branch, it is expected that an as-yet-unidentified immune receptor detects fungal infections and activates in turn the psh-dependent proteolytic cascade (Gottar, 2006).

GNBP1 belongs to the family of GNBP/β-glucan recognition proteins (βGRP). Members of this family have been reported to bind to β-(1,3)-glucan, a major component of the fungal cell wall. In Drosophila, three members of this family, GNBP1 to GNBP3, have been described. Among these, GNBP3 shows the greatest degree of similarity to lepidopteran β-(1,3)-glucan recognition proteins and was therefore a good candidate for a fungal-specific sensor. This paper reports that GNBP3 is indeed required for Toll pathway activation in response to fungal infections. Strikingly, it was also found that psh is required in a distinct yet complementary detection pathway that can be activated by fungal virulence factors (Gottar, 2006).

The detection of infections is a crucial step in the timely initiation of an appropriate immune response. In Drosophila, the use of nonentomopathogenic bacteria such as M. luteus and E. coli has allowed the delineation of both intracellular signal transduction pathways as well as the identification of five innate receptors (PRRs), PGRP-LC and PGRP-LE for the IMD pathway and PGRP-SA/GNBP1/PGRP-SD for the Toll pathway. To elucidate the mechanisms involved in the detection of fungi, concentration was placed on a somewhat artificial infection system using an opportunistic human pathogenic yeast, C. albicans. Understanding of the system was refined by using the entomopathogenic fungi B. bassiana and M. anisopliae (Gottar, 2006).

GNBP3 was found to be a PRR dedicated to the detection of fungi because (1) recombinant GNBP3 is able to bind in vitro to Candida and to polymeric chains of β-(1,3)-glucan; (2) it is required for the activation of the Toll pathway by polysaccharides of the fungal cell wall; (3) GNBP3 is required for resistance against yeast infections, including C. albicans, C. glabrata, and C. tropicalis, and against mold infections such as B. bassiana, M. anisopliae, and A. fumigatus; (4) GNBP3 triggers an adequate immune response; namely, it activates the antifungal Toll pathway in a spz-dependent manner. The possibility that another fungal receptor acts together with GNBP3 to activate the Toll antifungal host defense cannot be excluded (Gottar, 2006).

Of note is that fungi can induce the IMD pathway with short-term kinetics (Lemaitre, 1997). This induction was found to be dependent on PGRP-LC and not on GNBP3. One possibility is that a PGRP-LC coreceptor senses fungal microbial patterns. Alternatively, fungal cell wall constituents might bind directly to PGRP-LC. Interestingly, a coleopteran PGRP is able, in addition to its liaison to PGN, to bind with high affinity to tetralaminariose, a tetramer of β-(1,3)-glucan (Gottar, 2006).

As is the case for members of the PGRP family, the GNBP/βGRP proteins have evolved to recognize distinct carbohydrate chains that form the cell wall of microorganisms. Given their distribution in the arthropod lineage, it is likely that these two families form an essential part of their immunity repertoire. Whereas PGRP homologs exist in mammals, βGRP members have not been reported in vertebrates. However, the phagocytic and signaling receptor Dectin-1 detects β-(1,3)-glucans and may to some extent fulfill in mammals a primary function that is similar to that of GNBP3 in insects, i.e., the sensing of fungal infections (Gottar, 2006).

Because spz is required for Toll activation by GNBP3, it is proposed that the binding of GNBP3 to its microbial ligand leads to the activation of a proteolytic cascade that ultimately processes proSPZ into a functional Toll ligand. Because psh and GNBP3hades have distinct phenotypes as regards Toll pathway activation, and because the double mutant psh;GNBP3hades displays a stronger phenotype than either mutant alone when challenged with live fungi, PSH cannot belong exclusively to a proteolytic cascade activated by GNBP3. However, epistatic analysis reveals that the spz-dependent expression of Drosomycin induced by GNBP3 overexpression partly requires psh function. Taken together, these data indicate the existence of an alternative, psh-independent proteolytic cascade that mediates the GNBP3-dependent maturation of the Toll ligand Spätzle. This cascade is distinct from the one that activates Toll signaling during early embryogenesis (Gottar, 2006).

It is hypothesized that at least four distinct proteolytic cascades converge to process the Toll ligand Spätzle (SPZ). Dorsoventral (D/V) patterning occurs during early embryogenesis and involves the proteases Gastrulation Defective (GD), Snake (SNK), and Easter (EA): this proteolytic cascade is unlikely to be involved in the activation of Drosomycin expression by fungi. In addition to sensing virulence factors, PSH might function downstream of an unknown pattern recognition receptor (PRR). Indeed, epistatic analysis indicates that PSH partially functions downstream of GNBP3. PGN, peptidoglycan; SPE, Spätzle processing enzyme (Gottar, 2006).

An unexpected finding of this study is that the Toll pathway is normally induced in GNBP3hades mutants undergoing a B. bassiana infection. Yet, these mutants are more susceptible to this pathogen than wild-type flies. These observations suggest that GNBP3 fulfills other functions required in the host defense against fungal pathogens that are independent of its role in triggering the Toll pathway. Indeed, some biochemical evidence is available that GNBP3 is involved in other aspects of host defense (Gottar, 2006).

Many pathogens have adapted to their hosts and developed specific strategies to defeat their defenses. Fungi such as B. bassiana and M. anisopliae are able to infect insects following deposition of spores on the surface of the cuticle. To penetrate this physical barrier, they secrete several virulence factors such as chitinases and proteases. The PR1A protease is able to activate Drosomycin expression in the absence of infection when overexpressed in flies. This effect on Toll pathway activation is specific because it can be blocked in a psh background and depends on the proteolytic activity of PR1A. These data establish the proof of concept that a virulence factor can be detected by the innate immune system. Interestingly, the data indicate that PR1 can directly process PSH into its active form (Gottar, 2006).

PR1A is one of ten proteases in this subtilisin family and is expressed only during cuticle penetration. A PR1A/PR1B-deficient strain is still able to induce Drosomycin expression in a GNBP3hades mutant background, presumably through other fungal PR1 proteases. Thus, further work will be required to understand the multiple pathogenic mechanisms taking place during a natural fungal infection (Gottar, 2006).

The data show that the detection of fungal infections relies on a two-pronged sensor system that constitutes a partially redundant recognition system. The psh;GNBP3 double mutant strain consistently yields a stronger phenotype than that of the respective single mutants. Since only GNBP3 is strictly required in the defense against opportunistic yeasts, it is likely that the recognition of fungal patterns represents an ancestral, basal mode of infection sensing. The psh-dependent system that monitors virulence factors may have evolved secondarily in response to the selective pressure exerted by entomopathogenic fungi. Indeed, if the psh-based and the GNBP3-based sensing systems were perfectly redundant, it would be expected that the deletion of one of these systems would not prevent the activation of the Toll pathway. This is indeed what was observed when infecting flies with live C. albicans or with M. anisopliae. In contrast, Drosomycin inducibility is abolished in psh mutants, but not in GNBP3 mutants, infected by B. bassiana. These data indicate that B. bassiana has evolved a strategy that allows it to escape or to block GNBP3 surveillance (Gottar, 2006).

Future studies will reveal whether or not similar systems of virulence factor detection exist also to sense infection by entomopathogenic bacteria (Gottar, 2006).

It is surmised that some pathogens have developed strategies to inactivate the GNBP basal sensor system of Drosophila and that this led to the selection of a novel host counterstrategy: the surveillance of virulence factor activity. This theme is a central tenet of the current understanding of plant innate immunity. In plants, basal sensor systems detect the presence of microbial elicitors and trigger an immune response. Some virulence factors of the plant pathogen inhibit the elicitor-induced signaling by manipulating host proteins that regulate the host basal response. In some plant cultivars, a surveillance system based on R proteins 'guards' the targets of virulence factors (coded by microbial avirulence [avr] genes) and triggers a strong immune response when under attack. One example is provided by Arabidopsis, in which the cleavage of the endogenous PBS1 kinase by the Pseudomonas syringae type III effector AvrPphB, a cysteine protease, leads to the activation of the hypersensitive response by the R protein RPS5. A case possibly more relevant to fly immunity is provided by the tomato, in which the host protease Rcr3 is required for the recognition of the pathogen virulence factor Avr2 by the Cf-2 transmembrane receptor (Gottar, 2006).

Fungal proteases secreted by entomopathogenic fungi have to cross the structurally invariant cuticular barrier of the insect host that thus conditions the type of proteolytic activity required to degrade the cuticular proteins. This phenomenon may have been exploited by Drosophila to detect entomopathogenic infections in a mechanism that is hence conceptually related to the guard hypothesis of plants, although in this case PSH would monitor indirectly a passive defense mechanism, the protection provided by the bodily armor. To date, the analysis of the immune response in Drosophila has been largely limited to the study of laboratory strains in a controlled environment. By analogy to plant-pathogen interactions that involve avr genes and their cognate plant R resistance genes, a major challenge for the coming years will be to determine if the insect-pathogen interactions in a natural environment involve several distinct virulence factors and their associated host detection systems (Gottar, 2006).

The discovery of a host sensor system dedicated to the detection of virulence factor activity begs the question of the relevance of such a system to mammalian innate immunity. It has been reported that virulence factors such as the cholesterol-dependent cytolysin or pertussis toxin are able to induce immune responses through TLR4. In these cases, the possibility remains open that TLR4 functions as a coreceptor needed for intracellular signal transduction and that the actual recognition is mediated by unknown receptors. A second class of interest is that of the protease-activated receptors. Indeed, PAR2 has been implicated in the induction of the HB2 defensin by bacterial proteases in epithelial cells. Similarly, Citrobacter rodentium induces the intestinal release of host proteases that activate the PAR2 receptor and subsequent colonic inflammation. Finally, virulence factors from Salmonella and Yersinia have been shown to inhibit NF-κB and MAPK signaling. Thus, it is legitimate to ask if receptors dedicated to the perception of virulence factor activity have been selected during the evolution of the mammalian innate immune system (Gottar, 2006).

Proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-kappaB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Three serine proteases have been discovered that mediate Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative bacteria binding protein 3 (GNBP3), a receptor for a major cell wall component beta-1,3-glucan of fungi, and their order of activation have not been characterized yet. This study identified three serine proteases that are required for Toll activation by beta-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, it was shown that injection of beta-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal beta-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation (Roh, 2009).

Recent genetic studies using either in vivo RNAi strategy or loss-of-function mutation have demonstrated that several Drosophila serine proteases, such as Persephone, Grass, and Spirit, function upstream of the Drosophila SPE during activation of Toll (Kambris, 2006). Furthermore, El Chamy (2008) demonstrated that Drosophila Grass functions as a signaling component required for both the detection of fungi by GNBP3 and the sensing of Gram-positive bacteria by peptidoglycan recognition protein PGRP-SA. Also, it was suggested that another Drosophila serine protease, Persephone, previously shown to be specific for fungal detection, was required for the sensing of proteases that are released by fungi and bacteria (El Chamy, 2008). This indicates that Drosophila Persephone defines a parallel proteolytic cascade activated by virulence factors in the hemolymph. However, Drosophila Grass and Persephone genes do not show any homology with Tenebrio MSP, which functions as an apical serine protease common to both Tenebrio GNBP3 and PGRP-SA/GNBP1 complex. These results suggest the existence of significant differences in the proteolytic cascades working upstream of Toll in Tenebrio and Drosophila. In addition to this diversity of serine proteases, the gene expression sites of Tenebrio AMPs are also different from those of Drosophila AMPs. The Tenebrio Tenecin 1 gene was strongly induced in hemocytes of the Tenebrio larvae, while Drosophila AMPs are mainly produced by the fat body. Taken together, the differences in the organization of the serine protease cascade, in the type of AMPs and in the site of AMP synthesis between Drosophila and Tenebrio could represent adaptation mechanisms to fight specific environmental pathogens or may be linked to differences in their physiology. Analyzing differences and similarities in the mechanisms used by insects to fight microbial infection should shed light on the evolution of the immune system in this important phylum (Roh, 2009).

The activated Tenebrio SPE converted both the 79-kDa Tenebrio pro-phenoloxidase and clip-domain serine protease homologue 1 zymogen to their matured forms to generate an active melanization complex. This complex, consisting of a 76-kDa Tenebrio-activated phenoloxidase and a 43-kDa serine protease homologue 1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Because Tenebrio SPE also participates in the activation of the Toll pathway by GNBP3 upon β-1,3-glucan sensing and by PGRP-SA upon Lys-type PG recognition, these results indicate that both the melanization and the Toll pathway immune modules are sharing a common serine protease cascade for the regulation of these two major innate immune responses (Roh, 2009).

In summary, these biochemical studies shed light on the molecular mechanism regulating the Toll pathway by fungi. This work supports a model in which bacterial Lys-type PG and β-1,3-glucan recognition activate a common proteolytic cascade involving three different serine protease zymogens that are sequentially processed. This three-step proteolytic cascade leads to the maturation of Spätzle and the activation of the Toll intracellular signaling that control the synthesis of AMPs. A greater understanding of these two cascades could also facilitate the development of novel kits to detect bacteria and fungi in clinical and food products rapidly and sensitively (Roh, 2009).

Cytokine Spatzle binds to the Drosophila immunoreceptor Toll with a neurotrophin-like specificity and couples receptor activation

Drosophila Toll functions in embryonic development and innate immunity and is activated by an endogenous ligand, Spatzle (Spz). The related Toll-like receptors in vertebrates also function in immunity but are activated directly by pathogen-associated molecules such as bacterial endotoxin. This study presents the crystal structure at 2.35-Å resolution of dimeric Spz bound to a Toll ectodomain encompassing the first 13 leucine-rich repeats. The cystine knot of Spz binds the concave face of the Toll leucine-rich repeat solenoid in an area delineated by N-linked glycans and induces a conformational change. Mutagenesis studies confirm that the interface observed in the crystal structure is relevant for signaling. The asymmetric binding mode of Spz to Toll is similar to that of nerve growth factor (NGF) in complex with the p75 neurotrophin receptor but is distinct from that of microbial ligands bound to the Toll-like receptors. Overall, this study indicates an allosteric signaling mechanism for Toll in which ligand binding to the N terminus induces a conformational change that couples to homodimerization of juxtamembrane structures in the Toll ectodomain C terminus (Lewis, 2013).

Interaction with Pelle, Tube and MyD88

Tube and the protein kinase Pelle transduce an intracellular signal generated by the transmembrane receptor Toll. This signal directs import of Dorsal into ventral and ventrolateral nuclei, thereby establishing dorsoventral polarity. Tube protein associates with the plasma membrane during interphase. Tube sequences required for signaling interact with Pelle in a yeast two-hybrid assay. Fusion of the Pelle catalytic domain to the transmembrane receptor Torso is sufficient to induce ventral fates; this activity is independent of Toll or Tube. Fusion of the Tube protein to Torso also induces ventral fates, but only in the presence of functional Pelle. A model is proposed wherein Tube activates Pelle by recruiting it to the plasma membrane, thereby propagating the axis-determining signal (Galindo, 1995).

The Tube protein can function in a novel way to enhance DL activity. In the absence of DL, or when DL is cytoplasmic, Tube is also found in the cytoplasm of transfected cells. But when DL is localized to the nucleus, so isTube. Tube can then function to enhance reporter gene expression, either by cooperation with DL or as a GAL4-tube fusion protein. Tube thus appears capable of acting both as a chaperon or escort for DL as it moves to the nucleus, and then as a transcriptional coactivator. The intracytoplasmic domain of Toll, specifically the region sharing homology with the interleukin-1 receptor, is sufficient to induce DL-Tube nuclear translocation (Norris, 1995).

A complex signal transduction pathway functions in the early Drosophila embryo to establish dorsal-ventral polarity. Activation of this pathway results in the nuclear transport of the protein dorsal (dl), a member of the rel/NF-kappaB family of transcription factors. Genetic studies have identified three intracellular components whose activities are required for activation of dl: Toll, a transmembrane receptor; pelle (pll), a serine/threonine protein kinase, and tube, a protein of unknown function. The activities of these proteins were examined when coexpressed in Drosophila Schneider cells. Coexpression of pll with dl enhances dl nuclear localization and results in a modest increase in transcriptional activity. However, when pll is coexpressed with a specific mutant derivative of Toll (TlNaeI), although not with wild-type Toll, a striking synergistic activation of dl is detected. Unexpectedly, coexpression of pll plus TlNaeI, in the absence of dl, results in a similar synergistic activation of a GAL4-tube fusion protein. Based on these and other results, a model is proposed in which pll receives a signal from activated Toll and phosphorylates tube, which then participates directly in dl activation. The C-terminal intracytoplasmic region of Toll contains a 200 residue IL-1R (type I interleukin-1 receptor) homologous domain that is essential for Toll activity, plus an extra 68 residues of unique sequence at the very C terminus. It has been shown that eletion of this unique region increases transcriptional activity of Dorsal in cotransfected Schneider cells, suggesting it plays an inhibitory function (Norris, 1996).

Biochemical interactions are described between recombinant Toll, Pelle and Tube that provide insights into early events in activation of the signaling cascade. A tertiary complex exists prior to activation. The Pelle-Toll complex is required to suppress that kinase activity of Pelle. Pelle binds directly to a region within the Toll intracytoplasmic domain, providing the first evidence that these two evolutionarily conserved molecules physically interact. Pelle contains an N-terminal putative regulatory domain, consisting largely of a region with significant similarity to the consensus death domain, and a C-terminal catalytic domain. To determine whether either (or both) of these regions are required for the interaction with the intracellular domain of Toll (Toll IC), the Pelle N terminus (Pelle-R) and C terminus (Pelle-C) were produced separately by in vitro translation, and then tests were carried out to see if these molecules could bind any of several GST-Toll IC derivatives. The results indicate that the Pelle N terminus is incapable of binding any of the GST-Toll derivatives, whereas the catalytic region binds efficiently to both GST-Toll and GST-Toll ICNae (a derivative lacking the C-terminal ID but containing the entire IL-1R homology region). The Toll C-terminal inhibitory domain (ID) is neither necessary nor sufficient for this interaction (Shen, 1998).

It is thought that upon activation Pelle is autophosphorylated, and that this prevents binding to Toll as well as Tube. Autophosphorylation occurs in the N-terminal, death-domain-containing region of Pelle, a region dispensable for binding to Toll but required for enzymatic activity. Pelle phosphorylates Toll, within the region required for Pelle interaction, but this phosphorylation can be blocked by a previously characterized inhibitory domain at the Toll C terminus. These and other results allow for the proposal of a model by which multiple phosphorylation-regulated interactions between these three proteins lead to activation of the Dorsal signaling pathway (Shen, 1998).

It is proposed that the intracytoplasmic IL-1R homology domain of the Toll receptor initially interacts directly with unphosphorylated Pelle, and that the Toll ID helps down regulate kinase activity. Tube is also recruited to the complex through its interaction with Pelle and/or Toll, forming the Toll/Pelle/Tube ternary complex (Pelle inactive). Binding of Spätzle to Toll induces Toll dimerization, and the Toll cytoplasmic domain is modified through conformational changes or proteolysis of the Toll inhibitory domain, allowing activation of kinase activity (Pelle active). Active Pelle then phosphorylates multiple substrates, including itself, Toll and Tube. This causes disruption of the Toll/Pelle/Tube complex, freeing Pelle to phosphorylate unknown downstream targets, eventually resulting in Cactus phosphorylation/degradation and Dorsal phosphorylation (e. g., by protein kinase A [PKA] and possibly Pelle itself] and nuclear translocation. Phosphorylated Tube may also translocate with Dorsal and function as a transcriptional coactivator (Shen, 1998).

The direct interactions described between unphosphorylated Pelle, Toll and Tube are consistent with the existence of a ternary complex at the plasma membrane. Pelle interacts with Toll via residues in its catalytic domain, and with Tube via Pelle's N-terminal death domain: both interactions can occur simultaneously. An important question is whether the ternary complex forms independent of signaling. Previous studies have shown that the artificial recruitment of Pelle or Tube to the plasma membrane can initiate the signaling pathway independent of ligand binding. But it is not clear whether it is recruitment to the membrane per se that results in activation, or the dimerization of the Torso fusion proteins employed in these previous studies. A possible mechanism for Pelle activation is simply dimerization, induced naturally, it is suggested, by conformational changes in the ternary complex that occur following ligand binding. How might such changes be induced? There is considerable indirect evidence suggesting that Toll molecules interact: an attractive model posits that ligand binding induces dimerization or even aggregation. It is suggested that this leads to activation of signaling, i.e., of Pelle activity, by either (or both) of two related mechanisms: (1) oligomerization of Toll receptors increases the local ternary complex concentration and hence Pelle concentration, thereby favoring Pelle dimerization and activation by simple mass action; (2) ligand-induced Toll self-association causes a conformational change in the intracytoplasmic domain such that the ID is displaced, thereby facilitating Pelle activation, again perhaps by dimerization. A speculative possibility is that the ID is actually cleaved upon activation. The product of the strong dominant gain-of-function allele Toll 10b, which contains a single C to Y change in its extracellular domain, has been found in a partially proteolyzed form, such that full-length Toll 10b is associated with a truncated form lacking most or all of its extracellular domain as well as likely sequences from the very C terminus, i.e., the ID. Perhaps relevant to this, a putative PEST degradation sequence is situated between the IL-1R homology region and the ID. It is intriguing that the structure of this truncated product is similar to mammalian IL-1RAcp, which functions in IRAK activation during IL-1 signaling. In any event, it is proposed that IL-1R homology domain interactions activate Pelle via the direct, phosphorylation-sensitive protein-protein interactions described in this paper (Shen, 1998).

A signaling pathway active on the ventral side of the Drosophila embryo defines dorsoventral polarity. A cell surface signal relayed by Toll, Tube and Pelle releases the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus; free Dorsal translocates into nuclei and directs expression of ventral fates. Using the yeast two-hybrid system and immunoprecipitation experiments, scaffolding and anchoring interactions were defined among the pathway components. Dorsal binds specifically to Tube, Pelle and Cactus, and the protein kinase activity of Pelle differentially regulates its interactions with Dorsal and Tube. Amino acids 47-345 oof Dorsal are sufficient for interaction with both Tube and Pelle. This same region, the Rel homology domain, is also required for dimerization, for DNA binding and for interaction with Cactus. The Dorsal Rel domain is both necessary and sufficient for generation of a dorsoventral nuclear concentration gradient. Pelle and Dorsal interact with two separable domains of Tube. Pelle binds to the amino-terminal region of Tube that spans residues 25 to 173. Dorsal binds a C-terminal domain of Tube (amino acids 257 to 462). This region contains five copies of an evolutionarily conserved, 8-amino-acid repeat and is required for full Tube function. Interaction with Dorsal requires full-length Pelle. In contrast, only about 100 amino acids of Pelle (residues 26 to 129) are necessary and, most likely, sufficient for interaction with Tube. Pelle catalytic activity modulates its interaction with Dorsal and Tube. Drosophila Filamin (an Actin binding protein that localizes to the inner surface of the cell membrane) is identified as a potential adaptor linking the interaction network, via Tube, to the transmembrane receptor Toll. The Toll/IL-1 receptor homology appears to be both necessary and sufficient for the interaction of Toll with Filamin. The studies reported here have defined minimal interactions for Pelle (residues 26-129) and Tube (residues 25-173) that correspond closely to regions with similarity to a consensus death domain (see Reaper). Death domains have been identified in pathways regulating apoptosis, but their participation in the dorsoventral signaling cascade suggests a more general role in protein interactions mediating signal transduction (Edwards, 1997).

The Toll pathway recruits Tube and Pelle to the plasma membrane, a function that is required for the transmission of information from activated Toll receptors to the Dorsal-Cactus complex. An mRNA microinjection assay has demonstrated that targeting of either Tube or Pelle to the plasma membrane by myristylation is sufficient to activate the signal transduction pathway that leads to Dorsal nuclear translocation. Using confocal immunofluorescence microscopy it has also been shown that activated Toll induces a localized recruitment of Tube and Pelle to the plasma membrane. Together, these results strongly support the hypothesis that intracellular signaling requires the Toll-mediated formation of a membrane-associated complex containing both Tube and Pelle (Towb, 1998).

Tube and Pelle have been shown to signal constitutively when fused to the extracellular and transmembrane domains of the Drosophila Torso receptor. It has been proposed that constitutive activation of these chimeras is the direct result of membrane localization. However, the Torso sequences could also have activated the chimeras by (for example) mediating an ectopic extracellular protein-protein interaction. To determine whether membrane association is in fact sufficient to constitutively activate Tube and Pelle, Src90, the amino-terminal 90 amino acids of the Drosophila Src tyrosine kinase, was fused to full-length Tube and to the catalytic domain of Pelle. The Src 90 domain contains a myristylation signal that directs association with the plasma membrane. The src90-pelle and src90-tube fusion constructs were transcribed in vitro and then assayed their activity by microinjecting the resulting RNAs into syncytial blastoderm embryos. The ability of microinjected RNA transcripts to stimulate dorsoventral signaling was analyzed by examining cell fate markers in the cuticle secreted by the developing embryo. In wild-type embryos, Toll-mediated signaling to Dorsal leads to the formation of filzkörper and ventral denticle belts (cuticle structures representative of dorsolateral and ventral ectodermal fates, respectively). Mutations exist in which intracellular signaling is abolished due to a block in the extracellular pathway required for Toll ligand activation. For example, in embryos generated by females lacking function at the gastrulation defective locus (gd2 embryos), Toll is inactive and Dorsal remains exclusively cytoplasmic. Such embryos fail to form filzkörper and ventral denticles. Injection of either SRC90-TUBE or SRC90-PELLE mRNA into gd2 embryos directs signaling at the posterior site of microinjection, as evidenced by the appearance of filzkörper. SRC90-PELLE mRNA also induces the ectopic expression of ventral denticle belts. These results demonstrate the ability of both Src90-Tube and Src90-Pelle to direct Dorsal nuclear translocation in the absence of Toll activation. It is concluded that Pelle and Tube are activated upon targeting to the plasma membrane (Towb, 1998).

In the embryo, the majority of the Tube protein molecules associates with the cell surface and this association occurs over the entire circumference of the embryo. Since this localization is not restricted to ventral regions, it cannot represent an activated state of Tube. However, based on the Src90 fusion experiments, it is envisioned that Toll activates Tube by driving association of Tube with the plasma membrane. To detect any such signal-related membrane association, a detailed examination of Tube localization was carried out, using the nuclear concentration gradient of Dorsal as a marker for the dorsoventral axis. Surface views of wild-type embryos reveal that Tube forms a mesh-like array. Such a localization pattern is characteristic of proteins associated with the membranes and cytoskeleton that cap and surround each blastoderm nucleus. For Tube, the mesh-like pattern is most intense along the ventral midline, suggesting a concentration of Tube in this region. Confocal cross-sectional views confirm the existence of a dorsoventral asymmetry in the distribution of Tube at the embryo surface. Tube is more highly concentrated along the ventral surface than the dorsal surface; the difference in concentration is two-fold, as assayed by quantitation of staining intensity. Higher magnification ventral views reveal that Tube clusters along the embryo surface. These clusters or aggregates are approximately 2 mm in diameter and are located between adjacent ventral nuclei at sites of membrane invagination. On the dorsal side of the embryo, clusters of Tube are less readily detectable (Towb, 1998).

To determine whether the asymmetric distribution of Tube depends on Toll-mediated signaling, Tube localization was examined in genetic backgrounds that either inactivate or constitutively activate the signaling pathway. The gd2 mutation blocks Toll activation, whereas the Toll10b mutation alters the Toll extracellular domain so as to constitutively activate the receptor throughout the embryo. Both mutations eliminate the dorsoventral asymmetry in Tube staining observed in the wild type. However, Tube is more highly concentrated at the periphery of Toll10b embryos than gd2 embryos. The Toll10b and gd2 results support the model that activated Toll directs recruitment of Tube to the embryo surface. The asymmetry in Tube localization in the wild type is not, however, as striking as that observed for Cactus or Dorsal. Therefore, the distribution of Tube was also examined in P[Toll10b-bcd] embryos, in which Toll-mediated signaling is ectopically oriented along the anteroposterior axis. Females carrying the P[Toll10b-bcd] transgene express high levels of the Toll10b cDNA fused to the bicoid (bcd) 3'UTR and consequently produce embryos in which the bcd sequences localize the Toll10b mRNA to the anterior pole. Tube concentrates at the plasma membrane in and near the anterior pole of P[Toll10b-bcd] embryos. Moreover, clusters of Tube are prominent anteriorly on both the dorsal and ventral surfaces of such embryos. The Toll10b protein expressed from the P[Toll10b-bcd] construct also forms cell surface aggregates at the anterior pole. Comparison of the patterns of Tube and Toll localization in P[Toll10b-bcd] embryos indicates that the Toll aggregates colocalize with the Tube aggregates (Towb, 1998).

Since Tube and Pelle are thought to interact in embryos, it was reasoned that Pelle, like Tube, might localize to sites of Toll activation. Using a polyclonal anti-Pelle serum to stain wild-type embryos, it was found that Pelle is distributed throughout the embryo. A fraction of Pelle localizes to the surface of embryos, but there is no significant asymmetry in this distribution of Pelle across the dorsoventral axis. If Pelle undergoes a signal-dependent membrane association in wild-type embryos, this association is too short-lived or involves too few of the Pelle molecules in the embryo to be readily detectable. However, since localization studies had indicated that signal-dependent protein relocalization is enhanced in embryos expressing high levels of Toll10b protein at the anterior pole, Pelle localization was also examined in embryos generated by P[Toll10b-bcd] females. A large fraction of the Pelle protein localizes to the anterior end of P[Toll10b-bcd] embryos, indicating a significant recruitment or stabilization of Pelle protein at this pole. Confocal cross sections have revealed that the anterior-localized Pelle is predominantly at the embryonic periphery, possibly in association with the plasma membrane. High levels of activated Toll thus promote localization of both Tube and Pelle to the surface of the embryo. Since Tube localization at the anterior end of P[Toll10b-bcd] embryos mirrors that in the ventral portion of wild-type embryos, it is proposed that the localization of Pelle in P[Toll10b-bcd] embryos similarly reflects a relocalization occurring in wild-type embryos in response to Toll activation (Towb, 1998).

The products of four maternal genes, in addition to snake and easter, namely gastrulation defective (gd), nudel (ndl), pipe (pip), and windbeutel (wind), are required for processing of Spätzle to the Toll ligand. In the genetically ordered pathway for production of the Toll ligand, these genes act upstream of the snake gene and therefore are presumed to encode components required to activate the Snake protease. According to recent studies, the Pipe and Windbeutel proteins function during oogenesis to generate a spatially localized factor that defines the ventral side of the embryo. In current models, this factor is hypothesized to direct the ventral production of the Toll ligand by spatially restricting the activation of Snake and Easter, perhaps via a mechanism involving the Nudel and GD proteins. Nudel, a large modular protein with a central serine protease domain, is functionally complex and apparently involved in several processes important for embryogenesis. The Nudel protease is required for cross-linking of the eggshell, in addition to its role in establishing the embryonic dorsoventral axis. This observation has raised the intriguing possibility that the Nudel protease is involved in creating an extracellular matrix structure necessary for activating the proteolytic cascade that produces the Toll ligand (Han, 2000).

The role of the GD protein in activating this proteolytic cascade has been particularly enigmatic. GD is suspected to have an important role in this process, in part because it has the structure of a regulated serine protease like Snake and Easter, with a C-terminal domain homologous to serine proteases. However, it has been unclear whether GD functions as a serine protease because of peculiarities of its primary structure, such as the absence of a catalytic serine in the conserved position, or a recognizable proteolytic cleavage site for zymogen activation. It also has been unclear whether GD functions during early embryogenesis, rather than oogenesis, and is therefore directly involved in activating the proteolytic cascade that produces the Toll ligand. In addition, the position of GD with respect to Nudel, Pipe, and Windbeutel in the pathway producing the Toll ligand has not been known. In this study the GD protein is shown to be a serine protease. Three other genes act to restrict GD activity to the ventral side of the embryo. The data support a model in which the GD protease catalyzes the ventral activation of the proteolytic cascade that produces the Toll ligand (Han, 2000).

The fact that gd mutant embryos can be rescued by injection of GD mRNA indicates that the GD protein can function during early embryogenesis and thus could have a direct role in producing the Toll ligand. The downstream proteases Snake and Easter likely function in the perivitelline space of the embryo. It is presumed that GD is also a component of this extracellular compartment, since it has a signal sequence and is efficiently secreted by transfected S2 cells. However, it was not possible to rescue mutant embryos by injecting the perivitelline space of gd mutant embryos with recombinant GD in S2 cell culture medium, perhaps because of an insufficient amount of protein that also precluded extensive biochemical analysis (Han, 2000).

Genetic as well as biochemical evidence has been provided that GD functions as a serine protease. (1) The biological activity of GD RNA is destroyed by site-directed mutagenesis of putative catalytic serine and aspartic acid residues in GD. (2) A 29-kDa proteolytic fragment of recombinant GD protein reacts in vitro with affinity reagents specific for active serine proteases. The affinity labeling experiments also suggest that GD is synthesized as a zymogen that becomes activated by proteolytic cleavage, because a proteolytic fragment of GD, but not the full-length protein, reacts with the affinity reagents. GD lacks a recognizable zymogen activation site, so where cleavage normally occurs for GD to become an active serine protease is unclear. An active protease was generated by digestion of GD by trypsin, which is specific for basic residues, implying that GD could be normally activated by cleavage after a basic residue (Han, 2000).

The identification of GD as a serine protease raises the question, what is GD's substrate? A possible candidate is Snake, which is immediately downstream of GD in the Toll signaling pathway. The Snake zymogen may be activated by cleavage after a leucine residue and therefore by a chymotrypsin-like enzyme that prefers large hydrophobic residues. Whether GD has this specificity is unclear, since it diverges in sequence from other serine proteases in the region containing certain key residues that determine amino acid specificity. The fact that the 29-kDa form of GD reacts with affinity reagents designed for trypsin-like enzymes might suggest that GD is specific for basic residues. However, chymotrypsin also can cleave after basic residues, albeit inefficiently, and therefore could react with these affinity reagents under favorable conditions. Clearly, further biochemical experiments will be required to discern the amino acid specificity of the GD protease and to determine whether GD directly activates Snake (Han, 2000).

The RNA injection experiments reveal an important relationship between GD activity and Toll signaling. Injection of embryos with high concentrations of GD RNA causes more embryonic cells than normal to adopt ventral and lateral fates. A similar result has been observed in analogous experiments with spätzle RNA and with RNAs encoding 'preactivated' Snake and Easter but not the zymogen forms of these proteases. Thus, the level of GD activity may determine the amount of Spätzle processed to the Toll ligand, presumably by controlling the level of active Snake and Easter. In this case, an important function of Snake and Easter could be to amplify the signal generated by GD into the appropriate amount of Toll ligand necessary to establish the embryonic dorsoventral axis (Han, 2000).

These experiments revealed that gd acts downstream of nudel, pipe, and windbeutel, because the normal activity of each of these three genes is not required for injected GD RNA to induce ventral and lateral structures. After injection with a high level of GD RNA, embryos lacking maternal activity of nudel, pipe, or windbeutel develop a lateralized phenotype, which lacks the dorsoventral asymmetry of the ventralized phenotype seen when wild-type and gd mutant embryos are similarly injected. Thus, GD activity appears to be spatially uniform in the absence of normal Pipe, Windbeutel or Nudel function, implying that these three proteins are required to restrict GD activity to the ventral side of the embryo (Han, 2000).

How is GD activated? Although Nudel is the only known protease in the Toll signaling pathway that acts genetically upstream of GD, the data suggest that Nudel is not essential for GD protease activity. GD could be activated by an unidentified protease; alternatively, GD could self-activate. Proteases that initiate the protease cascades of blood clotting and apoptosis have a low level of enzymatic activity as zymogens, but become fully active upon a conformational change or upon oligomerization induced by a nonenzymatic cofactor. If GD is activated by a similar mechanism, then it should have a low level of protease activity as a zymogen (presumably below the sensitivity of detection in these affinity labeling experiments). In this case, the high concentration of GD protein produced by RNA injection into the embryo may have led to spontaneous GD activation that mimics and bypasses the normal activation mechanism. A model consistent with available data posits that the Nudel protease activates a cofactor, perhaps the ventral determinant provided by Pipe and Windbeutel, which in turn is necessary for activating the GD protease (Han, 2000).

Drosophila MyD88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of MyD88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative veorgrsion of MyD88 potently inhibits Toll-mediated signaling. MyD88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (FADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2001).

A BLAST search of the Drosophila genome identified the sequence encoding MyD88, a Drosophila homolog of human MyD88. Similar to its human homolog, Drosophila MyD88 contains an N-terminal death domain, an intermediate domain, and a TIR domain. However, unlike human MyD88, Drosophila MyD88 contains an additional 81 amino acids preceding the death domain and a 162-aa-long C-terminal region following the TIR domain (Horng, 2001).

Transfection of MyD88 into Drosophila S2 cells potently induces a Drosomycin reporter gene but not an Attacin reporter gene. This preferential ability to induce an antifungal gene is similar to that of Toll 10b, a constitutively active form of Toll, and suggests that MyD88 may be a component of the Toll-Tube-Pelle-Cactus-Dif signaling pathway. Previous studies have demonstrated that Toll-mediated Drosomycin induction requires the nuclear translocation of Dif. Dif is normally retained in the cytoplasm by the IkappaB inhibitor Cactus and is released only in response to signal-dependent degradation of Cactus. To test whether MyD88-mediated Drosomycin induction also depends on Cactus degradation, a Cactus mutant was constructed that contains mutations of the conserved serine residues that, in mammalian IkappaB, are the targets of signal-dependent phosphorylation. A Cactus mutant inhibits Drosomycin induction by MyD88 and, as expected, by Toll. This result indicates that, similar to Toll, MyD88 regulates Drosomycin induction through the Cactus-dependent pathway (Horng, 2001).

For further analyses, various deletion mutants of MyD88 were generated. Two of the deletion mutants, one containing the TIR domain and the C-terminal domain (amino acids 237-537) and another containing the intermediate, TIR, and C-terminal domains (amino acids 176-537), activate the Drosomycin reporter weakly (10-fold) in comparison to full length MyD88, indicating that the intact protein is required for optimal activity. However, the fact that these truncation mutants can still induce signaling is surprising, since they lack the death domain that mediates interactions with downstream signaling components. Moreover, similar analyses of human MyD88 have shown that a combination of the death domain and the intermediate domain is sufficient to induce signaling activity comparable to that of the wild-type protein. An equivalent truncation of dMyD88 (amino acids 1-237) retains no residual activity despite being well expressed, suggesting that there are some differences in domain function between human and Drosophila MyD88 proteins (Horng, 2001).

To determine whether MyD88 is a component of the Toll signaling pathway, attempts were made to identify a deletion mutant that would have dominant-negative activity. Therefore, three MyD88 deletion mutants that do not activate the Drosomycin reporter were tested for their ability to inhibit Toll-mediated Drosomycin induction. The strongest inhibitor was the death domain- and middle domain-containing construct (amino acids 1-237), which at low concentrations potently inhibits Toll-mediated Drosomycin induction in a dose-dependent manner (Horng, 2001).

To order MyD88 in the pathway with respect to Pelle, MyD88 was tested for its ability to be inhibited by PelleN, a dominant-negative form of Pelle that consists of the N-terminal death domain-containing region of Pelle. MyD88, like Toll, is strongly inhibited by PelleN. MyD88, however, does not inhibit Pelle, demonstrating that, similar to the mammalian pathway, MyD88 functions upstream of Pelle (Horng, 2001).

To further establish MyD88 as a component of the Toll pathway, whether MyD88 interacts with Toll was tested by coimmunoprecipitation assays. The TIR domain-containing MyD88 construct is detected in anti-Toll immunoprecipitates. Interestingly, when cotransfected with Toll 10b, MyD88 reproducibly appears as two distinct bands -- a slower migrating upper band that may correspond to phosphorylated MyD88 construct and a faster migrating lower band. The predominant form of MyD88 detected in immunoprecipitates is the faster migrating species. MyD88 therefore associates with Toll, presumably through TIR domains, and is a component of the active receptor complex (Horng, 2001).

Because human MyD88 associates with IRAK through death domains, a likely immediate downstream target of MyD88 is the IRAK homolog Pelle. Interaction between the death domain-containing dMyD88 construct (amino acids 1-237) and Pelle was examined. MyD88 is detected in Pelle immunoprecipitates, indicating that MyD88 interacts with Pelle, presumably through their death domains (Horng, 2001).

These results therefore demonstrate that MyD88 is an adaptor in the Toll signaling pathway downstream of the receptor and upstream of Pelle. From genetic analyses, the adaptor protein Tube has also been implicated to be downstream of Toll and upstream of Pelle in the Toll signaling pathway. The death domain of Tube also interacts with Pelle. Because Tube and MyD88 also contain death domains that could potentially mediate their interaction, tests were performed for association between these two proteins in immunoprecipitation assays; Tube and MyD88 do indeed interact. Therefore, MyD88 and Tube both function as adaptors downstream of Toll, exist in the same active complex along with Pelle, and are probably both involved in the recruitment and/or activation of Pelle. Understanding functional differences between these two adapters will require further analysis (Horng, 2001).

To identify other potential downstream targets of MyD88, a search of the Drosophila genome was performed for other sequences that encode death domain-containing proteins that may interact with MyD88. One such sequence encodes a protein with a death domain as well as a death effector domain and appears to be a homolog of mammalian FADD. This cDNA has been identified and named FADD (Hu, 2000). Whether FADD can interact with MyD88 was tested. Lysates from S2 cells transfected with MyD88 were incubated with anti-Flag beads to immunoprecipitate FADD, and immunoprecipitates were blotted with anti-V5 antibody to look for associated MyD88. A strong band corresponding to MyD88 was observed, indicating that MyD88 can interact with FADD through death domains. Overexpression of FADD in S2 cells, however, does not lead to activation of either the Drosomycin or Attacin reporters (Horng, 2001).

Mammalian FADD is recruited to the tumor necrosis factor receptor complex through homophilic death domain interactions with the adapter TNFR-associated death domain-containing protein (TRADD). In turn, FADD recruits procaspase-8 through homophilic death effector domain associations. It is speculated that Drosophila FADD may likewise recruit a Drosophila caspase to the Toll receptor complex. A potential candidate caspase is Dredd, an apical caspase with a long prodomain shown to be essential for induction of antibacterial genes. Indeed, analysis of immunoprecipitated lysates from cells cotransfected with Drosophila FADD, and either full length Dredd or the death effector domain of Dredd showed strong association of Dredd with FADD. A second study (Hu, 2000) has also shown interaction of dFADD with Dredd (Horng, 2001).

Thus Drosophila MyD88 is an adapter in the Toll signaling pathway. MyD88 associates with both Toll and Pelle and functions upstream of Pelle. Tube is known from genetic studies to be an adapter in the Toll pathway that functions upstream of Pelle. Why Toll should signal through MyD88 and Tube, two receptor-proximal adapters with seemingly similar functions, is not yet clear. MyD88 associates with the receptor Toll as well as the downstream adapter FADD, which in turn interacts with the apical caspase Dredd. Because caspases are essential executioners of the apoptotic machinery in organisms from nematodes to mammals, and because Dredd has been shown to be involved in apoptosis during Drosophila development, it is possible that Toll-1 or some of the other eight Tolls that exist in Drosophila may induce apoptosis (or another Dredd-dependent pathway) through the MyD88/dFADD/Dredd pathway in a cell-type specific and/or developmental stage-specific manner. The pathway comprised of Toll, MyD88, dFADD, and Dredd would be the first description of a pathway in invertebrates that links a cell surface receptor to an apical caspase. Such a pathway, if it exists, would enable extracellular stimuli, perhaps ligands secreted by other cells during development or pathogen-derived products during infection, to instruct invertebrate cells to undergo cell death. In addition, the Toll/MyD88/dFADD/Dredd pathway is remarkably similar to that activated by the receptors of the tumor necrosis factor receptor (TNFR) superfamily in mammals, in which FADD-mediated recruitment of caspase-8 leads to induction of apoptosis. Since the Drosophila genome does not encode any cell surface receptors homologous to TNFRs, it appears that the Toll/MyD88/dFADD/Dredd pathway is the evolutionary ancestor of the mammalian death receptor pathways. This possibility is further supported by the recent finding that human TLR2 can induce apoptosis through the MyD88/FADD/Caspase-8 pathway (Horng, 2001).

DmMyD88 encodes the Drosophila homolog of mammalian MyD88. DmMyD88 combines a Toll-IL-1R homology (TIR) domain and a death domain. Overexpression of DmMyD88 is sufficient to induce expression of the antifungal peptide Drosomycin, and induction of Drosomycin is markedly reduced in DmMyD88-mutant flies. DmMyD88 interacts with Toll through its TIR domain and requires the death domain proteins Tube and Pelle to activate expression of Drs, which encodes Drosomycin. DmMyD88-mutant flies are highly susceptible to infection by fungi and Gram-positive bacteria, but resist Gram-negative bacterial infection much as do wild-type flies. Phenotypic comparison of DmMyD88-mutant flies and MyD88-deficient mice shows essential differences in the control of Gram-negative infection in insects and mammals (Tauszig-Delamasure, 2002).

Weckle is a zinc finger adaptor of the toll pathway in dorsoventral patterning of the Drosophila embryo

The Drosophila Toll pathway takes part in both establishment of the embryonic dorsoventral axis and induction of the innate immune response in adults. Upon activation by the cytokine Spätzle, Toll interacts with the adaptor proteins DmMyD88 and Tube and the kinase Pelle and triggers degradation of the inhibitor Cactus, thus allowing the nuclear translocation of the transcription factor Dorsal/Dif. weckle (wek) has been identified as a new dorsal group gene that encodes a putative zinc finger transcription factor. However, its role in the Toll pathway was unknown. This study isolated new wek alleles and demonstrated that cactus is epistatic to wek, which in turn is epistatic to Toll. Consistent with this, Wek localizes to the plasma membrane of embryos, independently of Toll signaling. Wek homodimerizes and associates with Toll. Moreover, Wek binds to and localizes DmMyD88 to the plasma membrane. Thus, Wek acts as an adaptor to assemble/stabilize a Toll/Wek/DmMyD88/Tube complex. Remarkably, unlike the DmMyD88/tube/pelle/cactus gene cassette of the Toll pathway, wek plays a minimal role, if any, in the immune defense against Gram-positive bacteria and fungi. It is concluded that Wek is an adaptor to link Toll and DmMyD88 and is required for efficient recruitment of DmMyD88 to Toll. Unexpectedly, wek is dispensable for innate immune response, thus revealing differences in the Toll-mediated activation of Dorsal in the embryo and Dif in the fat body of adult flies (Chen, 2006).

Through a BLAST search, three zinc finger-containing genes, CG17568, CG10366, and CG6254, were identified as wek paralogs in the Drosophila genome. In addition to having high homology in the C-terminal zinc finger motifs, Wek also shows homology of 58%-65% and identity of 30%-43% in the N terminus with these three genes. This region is referred to as the WekN domain (amino acids 1-103) and the C-terminal region as the WekC domain (amino acids 273-470) that contains the six zinc fingers. The rest of Wek was designated as WekM domain (amino acids 104-272) . Although WekC shows high homology with several zinc finger proteins in mammals, no clear wek ortholog was identified in mammals using WekN and WekM (Chen, 2006).

Interaction between the TIR domains of Toll and DmMyD88 has been suggested by coimmunoprecipitation experiments in cultured cells. However, no interaction has been detected between DmMyD88 and the Toll intracytoplasmic domain in yeast two-hybrid assays, although interaction is detected between DmMyD88 and Tube. In addition, overexpression of the TIR domain of DmMyD88 leads to strong activation of the pathway, instead of behaving like a dominant negative, as one would expect for a domain-mediating interaction with upstream components of the pathway. Thus, there are indications that the situation may be more complex than initially assumed and involve a supplementary factor in the receptor complex. This study describes a zinc finger protein that functions as an adaptor in the Toll pathway. The data clearly establish that efficient recruitment of DmMyD88 to Toll in the embryo requires Wek and that Wek is part of the Toll receptor complex. This model is supported by coimmunoprecipitations in Drosophila cultured cells, immunolocalization in embryos, and finally genetics, as embryos laid by wek mutant females exhibit similar phenotypes as other Toll pathway mutants. Furthermore, strong genetic interactions are detected between wek and other genes of the Toll pathway. Interestingly, it was noticed that Wek also interacts with Toll-9 and to a lesser extent Toll-5, but not with other members of the Toll family. Toll, Toll-9, and Toll-5 are the only members of the family that are able to activate the Toll pathway in tissue culture cells, and hence there is a perfect correlation between the capacity to interact with Wek and the activation of the pathway (Chen, 2006).

Because DmMyD88 associates with active Toll, the observation that Wek is required to localize DmMyD88 to the cell surface even when Toll is active indicates that the physical interaction between active Toll and DmMyD88 alone might not be stable. This together with a series of binding results leads to a proposal that, before Toll activation, Wek bridges Toll and the DmMyD88/Tube complex to assemble into a large Toll/Wek/DmMyD88/Tube presignaling complex on the membrane. Upon Toll activation, Toll can now simultaneously associate with both adaptors (DmMyD88 and Wek) to assemble a much more stable Toll/Wek/DmMyD88/Tube complex that leads to the differential association and activation of Pelle. This model not only explains why DmMyD88 is still membrane associated when Toll is inactive on the dorsal side of the embryo but also explains why Tube and DmMyD88 distribute asymmetrically along the dorsoventral axis with increased concentration along the ventral surface. In the absence of Wek, the weak association between active Toll and DmMyD88/Tube might either completely or partially abolish the recruitment and activation of Pelle to produce a range of dorsalized phenotypes. According to this model, active Toll interacts with a Spätzle dimer via its ectodomain and with a Wek dimer via its cytoplasmic tail. However, overexpression of Wek can not partially rescue the completely dorsalized phenotype of spz mutant embryos, indicating that Wek alone is not sufficient to mediate Toll activation (Chen, 2006).

Although the genetic and biochemical data support that the zinc finger-containing Wek acts at the plasma membrane as an adaptor during dorsoventral patterning, Wek preferentially accumulates in the nucleus of fat body cells. Thus, the possibility that Wek might have a nuclear function as well cannot be completely excluded. This would be reminiscent of Armadillo, which acts not only as an adaptor of Cadherin but also as a transcription factor in the nucleus (Chen, 2006).

Surprisingly, the data suggest that wek is not required for the Toll-mediated induction of the drosomycin gene in response to immune challenge. Indeed, wild-type responses were observed in the fat body of wek mutant flies, and in S2 cells depleted of wek mRNA by RNAi. This result might be explained by a different threshold level for wek function in development and immunity: the residual activity of the Toll pathway in weklor/wekEX14 transheterozygote flies, and in wek dsRNA-treated S2 cells, could be sufficient for the immune response. It is noted, however, that experiments with DmMyD88 revealed on the contrary that the dorsoventral patterning function appears to be less sensitive than the immune function, as flies homozygote for the hypomorphic allele DmMyD88EP2133 are severely impaired in their host-defense functions, but not for dorsoventral patterning. Thus, although a role of wek in Toll-mediated immune defenses cannot be formally rule out, the most likely explanation for the results is that induction of drosomycin expression by Toll in fat body and S2 cells does not depend on wek. This interpretation is supported by the fact that in the fat body Wek does not colocalize with Toll at the plasma membrane but rather preferentially localizes to the nucleus (Chen, 2006).

Differences between Toll signaling in the embryo and in adults have already been reported in several instances. The first difference pertains to the identity of the transcription factor induced, Dorsal in the embryo and Dif in adults. There is also convincing evidence that Toll not only signals to Cactus, but also induces phosphorylation of Dorsal. In addition, differences were observed in the interaction of Dorsal and Dif with cofactors. For example, unlike Dorsal, Dif does not appear to interact and synergize with basic-helix-loop-helix transcription factors or to be affected by the negative regulator WntD. Conversely, the coactivator dTRAP80 modulates activation of Dif, but not Dorsal, in S2 cells. Wek might therefore regulate a Dorsal-specific output of the Toll pathway. This hypothesis is, however, difficult to reconcile with the fact that Wek acts between two components of the pathway, Toll and DmMyD88, which are both required for activation of Dorsal in the embryo and Dif in adults. Furthermore, the proposed function of Wek as an adaptor that connects Toll and DmMyD88 does not explain why this factor is not required in adults and S2 cells. The most likely reason to explain this paradox is that another molecule substitutes for Wek in fat body and S2 cells. The three paralogs of wek (CG6254, CG17568, and CG10366) found in the Drosophila genome are prime candidates to carry this function and connect Toll to DmMyD88 in immune-competent cells. The first two contain both the N region and the zinc finger-containing C region, whereas the third one contains all three domains. However, RNAi-mediated silencing of these genes in S2 cells did not affect Toll signaling. Thus, the identity of the factor that bridges Toll and DmMyD88 in fat body cells remains unknown at this stage (Chen, 2006).

The different requirement for Wek in embryos and adults may also reflect the presence of Dorsal and Dif in the Toll receptor complex. Because the NF-κB-like molecules targeted by Toll are different in the embryo and in adults, such a mechanism could provide an explanation for the embryo-specific phenotype of wek mutant flies. In support of this hypothesis, Cactus-bound Dorsal has been shown to form a complex with Tube and Pelle, in which Cactus may be phosphorylated by Pelle. It is interesting to note that this hypothetic model of activation of Dorsal and Dif at the receptor complex is evocative of the activation of the transcription factor IRF7 by the kinase IRAK1 upon stimulation of TLR7 or TLR9 in mammalian cells (Chen, 2006).

beta-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. This study reports a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. These findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz (Tipping, 2010).

This study demonstrate that the Krz protein is necessary for setting a precise level of activation of two maternal signalling pathways, Torso and Toll. This activity of Krz helps to establish the correct domains of expression of developmental patterning regulators that are under the control of these pathways (Tipping, 2010).

Genetic and protein interaction data suggest a new mechanism by which Krz may limit the activity of Torso. It was observed that Krz preferentially binds and sequesters an inactive form of ERK, thereby making it unavailable for activation by the upstream kinases such as MEK. Such a mechanism of direct inhibition of ERK activation by β-arrestin binding has not been previously reported. This mechanism is consistent with the observed in vivo effects of loss of krz on ERK activity. In krz maternal mutant embryos, ERK is not sequestered and therefore more ERK is available to transduce Torso signals, resulting in hyperactivation of Torso target genes, tll and hkb. Furthermore, consistent with this model is the observation that Krz and MEK apparently compete for ERK when all three proteins are co-expressed in S2 cells (Tipping, 2010).

Interaction assays using mutated forms of Krz and ERK indicate that the conformations of both proteins have an effect on their binding affinity. On binding to an activated GPCR, the arrestin molecule undergoes a dramatic conformational change that can be mimicked by specific mutations (Gurevich, 2004). In immunoprecipitation experiments it was observed that such 'pre-activated' form of Krz (R209E) has a much greater affinity for ERK, compared with the wild-type Krz protein, and that this higher affinity is also observed for the equivalent mutant of human β-arrestin2. This suggests that the ERK-binding ability of β-arrestin may be affected by its conformation, but it is unknown at present whether any upstream signals convert Krz into an activated form in the embryo. Overexpression of Krz-R209E using the da-GAL4 driver did not result in any observable phenotype and could rescue zygotic loss of krz, suggesting that it retains most of the functions of wild-type Krz (data not shown) (Tipping, 2010).

It was observed that the conformation of ERK itself has a large effect on its interactions with Krz. In the binding experiments, activated forms of ERK bind Krz (and human β-arrestin2) with lower affinity, compared with wild-type inactive ERK. Moreover, mutations in the TEY motif, which render ERK constitutively inactive, also lower its affinity for Krz, which is at a first glance a surprising result. However, previous studies have shown that both types of mutations in the TEY motif, which is a part of the activation loop, increase disorder in the lip region and cause a conformational change in the ERK molecule that makes it different from the basal state. It is therefore speculated that the activation loop may be involved in mediating an interaction of ERK with β-arrestin. Consistent with the current results, deviation of ERK structure from the basal state would decrease its association with β-arrestin (Tipping, 2010).

Other studies have reported formation of protein complexes containing β-arrestins and an activated form of ERK. It is possible that in those experimental conditions other binding partners, such as Raf or the activated receptor, assist in stabilizing the complex of MAP kinases with β-arrestin. This study has shown that although Krz can bind to the Drosophila homologues of both MEK and Raf, overexpression of Krz does not increase production of dpERK by the MAPK cascade downstream of activated RTKs, but instead appreciably inhibits it in the absence of overexpressed Raf. The data do not rule out a possibility that Krz may still promote ERK activation in other biological contexts, particularly downstream of activated GPCRs, but this question awaits further investigation (Tipping, 2010).

Interestingly, the sequestration mechanism of ERK inhibition described in this study is different from the effects of Krz on Notch. Previous studies have shown that Krz inhibits Notch activity by forming a ternary complex with Deltex and the Notch receptor. Formation of this complex increases Notch turnover and thereby downregulates Notch signalling (Mukherjee, 2005). No change was observed in ERK turnover in the presence of wild-type overexpressed Krz, suggesting that Krz is unlikely to be involved in the regulation of ERK stability. However, given the versatility of molecular functions displayed by β-arrestins, it is possible that there are other, as yet uncharacterized mechanisms by which Krz controls signalling downstream of RTKs (Tipping, 2010).

The inhibitory effects of Krz on ERK activity are not limited to the Torso pathway and early embryogenesis, but are also observed in other tissues and at later developmental stages. Thus, broadening of the dpERK patterns activated by EGFR and Btl was observed in krz maternal mutant embryos. An increase in the overall levels of dpERK during mid-to-late embryogenesis was also detected on western blots. Later in development, ERK is activated by EGFR in the wing and both EGFR and Sevenless in the eye. Genetic data suggest that Krz also inhibits ERK activity in these tissues during larval development. A broad involvement of Krz in inhibiting ERK activity suggests that Krz has a general inhibitory role to limit the activity of different RTKs in Drosophila development (Tipping, 2010).

In addition to its effects on RTK signalling, it was observed that Krz has an important role in limiting the activity of the Toll receptor, which specifies the development of the ventral structures. Other studies have reported that mammalian β-arrestins can downregulate NF-κB signalling by binding and stabilizing the NF-κB inhibitor IκBα. The inhibitory effects of Krz on Dorsal may involve a similar mechanism. It was observed that Krz can directly bind to the Drosophila orthologue of IκBα, Cactus, suggesting that the mechanism of NF-κB inhibition by β-arrestins at the level of IκBα may be conserved. Consistent with this finding, a decrease was detected in the level of the Cactus protein in krz maternal mutants at 0-4 h of development, which may explain the observed expansion of the nuclear gradient of Dorsal in these mutants. It is still unclear why expansion of Dorsal nuclear localization is more pronounced in the posterior half of the embryo (Tipping, 2010).

In the developing embryo, the Torso and Toll pathways do not work in isolation, but are involved in cross-regulatory interactions on certain common targets, such as zen. zen is repressed by nuclear Dorsal in the ventral part of the embryo, and relieved of this repression (de-repressed) by the signalling activity of Torso emanating from the embryo poles. The molecular mechanism of this de-repression is still unknown. It was observed that loss of krz shifts the balance of the effects of Torso on Toll, which results in an inappropriate expansion of zen expression at the embryo poles. It is speculated that Krz helps Torso to achieve a precise level of de-repression of zen by limiting the activity of ERK. Krz is thus able to control the separate activities of the Torso and Toll pathways (reflected in its effects on tll, hkb, twi, and rho), as well as regulate common Torso and Toll targets such as zen. For such pathways that are engaged in cross-regulatory interactions, Krz ensures that a proper level of signalling activity from one pathway reaches the other. This function adds an important new mechanism to understanding of the ways in which signalling pathways are coordinately regulated during development (Tipping, 2010).

A ubiquitous distribution of Krz in the embryo agrees with the dysregulation of multiple pathways observed in krz mutant animals. As overexpression of Krz does not cause any obvious defects, the level of Krz itself is not limiting for the regulation of signalling. Instead, Krz apparently makes other signalling co-factors limiting for their respective pathways, essentially working as a molecular 'sponge' to prevent pathway hyperactivity. Specificity of Krz function is likely to be determined by its selective interactions with specific pathway co-factors. Maternal loss of krz function thus affects multiple developmental signalling pathways, resulting in an accumulation of defects that ultimately lead to severe morphological abnormalities such as a disruption of gastrulation movements. By analysing the effects of loss of krz on individual pathways in vivo, this study has been able to show its role in the regulation of RTK and Toll signalling. Future studies will likely reveal other pathways and levels of regulation that are under the control of the Drosophila β-arrestin Krz (Tipping, 2010).

Persephone/Spatzle pathogen sensors mediate the activation of Toll receptor signaling in response to endogenous danger signals in apoptosis-deficient Drosophila

Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially lead to necrosis, resulting in the release of endogenous immunogenic molecules such as DAMPs (damage-associated molecular patterns) and a non-infectious inflammatory response. However, the details about how danger signals from apoptosis-deficient cells are detected and translated to an immune response are largely unknown. This study found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous Toll ligand Spatzle (Spz). It is speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. It was demonstrated that Toll signaling activation required the action of Persephone, a CLIP-domain serine protease that usually reacts to microbial proteolytic activities. These findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic responses in apoptosis-deficient Drosophila mutants (Ming, 2014).

Identifying USPs regulating immune signals in Drosophila: USP2 deubiquitinates Imd and promotes its degradation by interacting with the proteasome

Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NFκB-dependent signals, which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, a focused RNA interference library was created and screened on Drosophila NFκB-like pathways Toll and Imd in cultured S2 cells, and the function of selected genes were further analysed in vivo. USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NFκB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. It was further shown that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments. This work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis (Engel, 2014).


Toll: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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