A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect

The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Sp?tzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Sp?tzle processing enzyme (SPE), a terminal enzyme that converts Sp?tzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Sp?tzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Sp?tzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Sp?tzle processing is essential for the PG-dependent Toll signaling pathway.     

Ligand-receptor and receptor-receptor interactions act in concert to activate signaling in the Drosophila toll pathway

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Sp?tzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Sp?tzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Sp?tzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.     

A family of proteins related to Spätzle, the toll receptor ligand, are encoded in the Drosophila genome

The Drosophila gene Sp?tzle encodes the activating ligand for the Toll receptor. This signaling pathway is required for dorso-ventral patterning in the early embryo and an antifungal immune response in larvae and adults. The genome sequence of Drosophila shows that there are a total of eight Toll-like receptors and these may function in other aspects of embryonic development and innate immunity. Here we describe five Drosophila homologues of Sp?tzle (Spz2-6) found using an iterative searching method. All five appear to encode proteins containing neurotrophin-like cystine-knot domains. In addition, most retain a characteristic intron-exon structure shared with the prototype Sp?tzle gene. This provides evidence that the family arose by ancient gene duplication events and indicates that the gene products may represent activating ligands for corresponding Toll receptors. Expression studies show that only Spz4 is expressed strongly in larvae and adults and thus may be involved in an ancillary antifungal response mediated by Toll-5. By contrast, Spz6 shows a complex spatial and temporally regulated expression pattern in the late embryo. Thus the new Toll/Sp?tzle families of signaling molecules may have important roles in other aspects of development and immunity.     

Regulation of Easter activity is required for shaping the Dorsal gradient in the Drosophila embryo

Dorsoventral polarity of the Drosophila embryo requires maternal sp?tzle-Toll signaling to establish a nuclear gradient of Dorsal protein. The shape of this gradient is altered in embryos produced by females carrying dominant alleles of easter (ea(D)). The easter gene encodes a serine protease that generates processed Sp?tzle, which is proposed to act as the Toll ligand. By examining the expression domains of the zygotic genes zen, sog, rho and twist, which are targets of nuclear Dorsal, we show that the slope of the Dorsal gradient is progressively flattened in stronger ea(D) alleles. In the wild-type embryo, activated Easter is found in a high M(r) complex called Ea-X, which is hypothesized to contain a protease inhibitor. In ea(D) embryo extracts, we detect an Easter form corresponding to the free catalytic domain, which is never observed in wild type. These mutant ea(D) proteins retain protease activity, as determined by the production of processed Sp?tzle both in the embryo and in cultured Drosophila cells. These experiments suggest that the ea(D) mutations interfere with inactivation of catalytic Easter, and imply that this negative regulation is essential for generating the wild-type shape of the Dorsal gradient.     

A Spätzle-processing enzyme required for toll signaling activation in Drosophila innate immunity

The Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Sp?tzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Sp?tzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Sp?tzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Sp?tzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.     

Spätzle regulates the shape of the Dorsal gradient in the Drosophila embryo

Dorsal-ventral polarity of the Drosophila embryo is established by a nuclear gradient of Dorsal protein, generated by successive gurken-Egfr and sp?tzle-Toll signaling. Overexpression of extracellular Sp?tzle dramatically reshapes the Dorsal gradient: the normal single peak is broadened and then refined to two distinct peaks of nuclear Dorsal, to produce two ventral furrows. This partial axis duplication, which mimics the ventralized phenotype caused by reduced gurken-Egfr signaling, arises from events in the perivitelline fluid of the embryo and occurs at the level of Sp?tzle processing or Toll activation. The production of two Dorsal peaks is addressed by a model that invokes action of a diffusible inhibitor, which is proposed to normally regulate the slope of the Dorsal gradient.     

Molecular Mechanism That Induces Activation of Sp?tzle,the Ligand for the Drosophila Toll Receptor

The Drosophila Toll receptor is activated by an endogenous cytokine ligand Spätzle. Active ligand is generated in response to positional cues in embryonic dorso-ventral patterning and microbial pathogens in the insect immune response. Spätzle is secreted as a pro-protein and is processed into an active form by the serine endoproteases Easter and Spätzle-processing enzyme during dorso-ventral patterning and infection, respectively. Here, we provide evidence for the molecular mechanism of this activation process. We show that the Spätzle prodomain masks a predominantly hydrophobic region of Spätzle and that proteolysis causes a conformational change that exposes determinants that are critical for binding to the Toll receptor. We also gather that a conserved sequence motif in the prodomain presents features of an amphipathic helix likely to bind a hydrophobic cleft in Spätzle thereby occluding the putative Toll binding region. This mechanism of activation has a striking similarity to that of coagulogen, a clotting factor of the horseshoe crab, an invertebrate that has changed little in 400 million years. Taken together, our findings demonstrate that an ancient passive defense system has been adapted during evolution and converted for use in a critical pathway of innate immune signaling and embryonic morphogenesis.     

Assembly of oligomeric death domain complexes during Toll receptor signaling

The Drosophila Toll receptor is activated by the endogenous protein ligand Sp?tzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling.     

Molecular control of phenoloxidase-induced melanin synthesis in an insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Sp?tzle-processing enzyme is a key enzyme in the production of cleaved Sp?tzle from pro-Sp?tzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Sp?tzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.     

Expression and regulation of Spätzle-processing enzyme in Drosophila

The Drosophila melanogaster Toll receptor controls embryonic dorsal-ventral axis formation and is crucial for the innate immune response. In both cases, Toll is activated by the enzymatically cleaved form of its ligand Sp?tzle (Spz). During axis formation, Spz is cleaved by the maternally provided serine protease Easter while the Sp?tzle-processing enzyme (SPE) activates Spz after infection. We confirm the role of SPE in immunity and show that it is a zygotic gene specifically expressed in immune tissues implying that the dual activation of Spz is achieved by differential spatiotemporal expression of two similar but distinct serine proteases.     

Biophysical characterization of refolded Drosophila Spätzle, a cystine knot protein, reveals distinct properties of three isoforms

The Drosophila Sp?tzle protein, involved in the embryonic development of the dorsal-ventral axis and in the adult immune response, is expressed as a proprotein and is activated by the serine proteinases Easter or Sp?tzle-processing enzyme. Proteolytic cleavage generates a 106-amino acid COOH-terminal fragment, C106, homologous to the mature form of nerve growth factor NGF, a cystine knot protein. Through alternative splicing, the Sp?tzle gene encodes for several isoforms that (with one exception, the "propeptide isoform") share C106 but differ in the prosequence. Three isoforms have been expressed recombinantly in Escherichia coli strains. The propeptide isoform could be expressed in soluble form and is unstructured according to CD and NMR measurements. Dimeric full-length Sp?tzle isoforms have been refolded from insoluble inclusion bodies and are able to rescue Sp?tzle-deficient embryos. Although the two full-length isoforms exhibit similar far-UV CD spectra, large differences in tryptophan fluorescence quenching by the respective pro-parts are observed. Both full-length isoforms exhibited highly cooperative folding transitions. Proteolytic digestion using trypsin resulted in C106, whose unfolding exhibits lower thermodynamic stability and cooperativity compared with the full-length proteins. The structure of C106 reveals a T-shaped dimer with significant differences to NGF and a deep internal cavity. Substantial beta-sheet formation is observed between the two monomers, whereas a long loop containing the single tryptophan residue is disordered in the crystals. Our results suggest that the propeptides stabilize the tertiary structure of the "mature" Sp?tzle cystine knot.     

Structural insight into the mechanism of activation of the Toll receptor by the dimeric ligand Spätzle

The Drosophila Toll receptor, which functions in both embryonic patterning and innate immunity to fungi and Gram-positive bacteria, is activated by a dimeric cytokine ligand, Sp?tzle (Spz). Previous studies have suggested that one Spz cross-links two Toll receptor molecules to form an activated complex. Here we report electron microscopy structures of the Toll ectodomain in the absence and presence of Spz. Contrary to expectations, Spz does not directly cross-link two Toll ectodomains. Instead, Spz binding at the N-terminal end of Toll predominantly induces the formation of a 2:2 complex, with two sites of interaction between the ectodomain chains, one located near to the N terminus of the solenoid and the other between the C-terminal juxtamembrane sequences. Moreover, Toll undergoes a ligand-induced conformational change, becoming more tightly curved than in the apo form. The unexpected 2:2 complex was confirmed by mass spectrometry under native conditions. These results suggest that activation of Toll is an allosteric mechanism induced by an end-on binding mode of its ligand Spz.     

Pillars article: the dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996. 86: 973-983

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, sp?tzle regulatory gene cassette, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.     

In vivo RNA interference analysis reveals an unexpected role for GNBP1 in the defense against Gram-positive bacterial infection in Drosophila adults

The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate that GNBP1 is required for Toll activation in response to Gram-positive bacterial infection; GNBP1 double-stranded RNA expression renders flies susceptible to Gram-positive bacterial infection and reduces the induction of the antifungal peptide encoding gene Drosomycin after infection by Gram-positive bacteria but not after fungal infection. This phenotype induced by GNBP1 inactivation is identical to a loss-of-function mutation in PGRP-SA, and our genetic studies suggest that GNBP1 acts upstream of the Toll ligand Sp?tzle. Altogether, our results demonstrate that the detection of Gram-positive bacteria in Drosophila requires two putative pattern recognition receptors, PGRP-SA and GNBP1.     

Self-organized shuttling: generating sharp dorsoventral polarity in the early Drosophila embryo

Morphogen gradients pattern tissues and organs during development. When morphogen production is spatially restricted, diffusion and degradation are sufficient to generate sharp concentration gradients. It is less clear how sharp gradients can arise within the source of a broadly expressed morphogen. A recent solution relies on localized production of an inhibitor outside the domain of morphogen production, which effectively redistributes (shuttles) and concentrates the morphogen within its expression domain. Here, we study how a sharp gradient is established without a localized inhibitor, focusing on early dorsoventral patterning of the Drosophila embryo, where an active ligand and its inhibitor are concomitantly generated in a broad ventral domain. Using theory and experiments, we show that?a sharp?Toll activation gradient is produced through "self-organized shuttling," which dynamically relocalizes inhibitor production to lateral regions, followed by inhibitor-dependent ventral shuttling of the activating ligand Sp?tzle. Shuttling may represent?a general paradigm for patterning early embryos. PAPERFLICK: