The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2.
ABSTRACT: Mutants of the necrotic (nec) gene in Drosophila melanogaster die in the late pupal stage as pharate adults, or hatch as weak, but relatively normal-looking, flies. Adults develop black melanized spots on the body and leg joints, the abdomen swells with hemolymph, and flies die within 3 or 4 days of eclosion. The TOLL-mediated immune response to fungal infections is constitutively activated in nec mutants and pleiotropic phenotypes include melanization and cellular necrosis. These changes are consistent with activation of one or more proteolytic cascades. The nec gene corresponds to Spn43Ac, one of a cluster of three putative serine proteinase inhibitors at 43A1.2, on the right arm of chromosome 2. Although serpins have been implicated in the activation of many diverse pathways, lack of an individual serpin rarely causes a detectable phenotype. Absence of Spn43Ac, however, gives a clear phenotype, which will allow a mutational analysis of critical features of the molecular structure of serpins.
Project description:We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles gambiae using phylogenetic, molecular, reverse genetic, and cell biological tools. The results strongly implicate SRPN6 in the innate immune response against Plasmodium. This gene belongs to a mosquito-specific gene cluster including three additional Anopheles serpins. SRPN6 expression is induced by Escherichia coli and both rodent and human malaria parasites. The gene is specifically expressed in midgut cells invaded by Plasmodium ookinetes and in circulating and attached hemocytes. Knockdown of SRPN6 expression by RNA interference in susceptible An. stephensi leads to substantially increased parasite numbers, whereas depletion in susceptible An. gambiae delays progression of parasite lysis without affecting the number of developing parasites. However, the An. gambiae SRPN6 knockdown increases the number of melanized parasites in the L3-5 refractory strain and in susceptible G3 mosquitoes depleted of CTL4. These results indicate that AsSRPN6 is involved in the parasite-killing process, whereas AgSRPN6 acts on parasite clearance by inhibiting melanization and/or promoting parasite lysis. We propose that these observed phenotypic differences are due to changed roles of the respective target serine proteases in the two mosquito species.
Project description:Serpins are a broadly distributed superfamily of protease inhibitors that are present in all kingdoms of life. The acronym, serpin, is derived from their function as potent serine proteases inhibitors. Early studies of serpins focused on their functions in haemostasis since modulating serine proteases activities are essential for coagulation. Additional research has revealed that serpins function in infection and inflammation, by modulating serine and cysteine proteases activities. The aim of this review is to summarize the accumulating findings and current understanding of the functions of serpins in host-pathogen interactions, serving as host defense proteins as well as pathogenic factors. We also discuss the potential crosstalk between host and pathogen serpins. We anticipate that future research will elucidate the therapeutic value of this novel target.
Project description:Serpins are a broadly distributed family of protease inhibitors that use a conformational change to inhibit target enzymes. They are central in controlling many important proteolytic cascades, including the mammalian coagulation pathways. Serpins are conformationally labile and many of the disease-linked mutations of serpins result in misfolding or in pathogenic, inactive polymers.
Project description:The humoral response to fungal and Gram-positive infections is regulated by the serpin-family inhibitor, Necrotic. Following immune-challenge, a proteolytic cascade is activated which signals through the Toll receptor. Toll activation results in a range of antibiotic peptides being synthesised in the fat-body and exported to the haemolymph. As with mammalian serpins, Necrotic turnover in Drosophila is rapid. This serpin is synthesised in the fat-body, but its site of degradation has been unclear. By "freezing" endocytosis with a temperature sensitive Dynamin mutation, we demonstrate that Necrotic is removed from the haemolymph in two groups of giant cells: the garland and pericardial athrocytes. Necrotic uptake responds rapidly to infection, being visibly increased after 30 mins and peaking at 6-8 hours. Co-localisation of anti-Nec with anti-AP50, Rab5, and Rab7 antibodies establishes that the serpin is processed through multi-vesicular bodies and delivered to the lysosome, where it co-localises with the ubiquitin-binding protein, HRS. Nec does not co-localise with Rab11, indicating that the serpin is not re-exported from athrocytes. Instead, mutations which block late endosome/lysosome fusion (dor, hk, and car) cause accumulation of Necrotic-positive endosomes, even in the absence of infection. Knockdown of the 6 Drosophila orthologues of the mammalian LDL receptor family with dsRNA identifies LpR1 as an enhancer of the immune response. Uptake of Necrotic from the haemolymph is blocked by a chromosomal deletion of LpR1. In conclusion, we identify the cells and the receptor molecule responsible for the uptake and degradation of the Necrotic serpin in Drosophila melanogaster. The scavenging of serpin/proteinase complexes may be a critical step in the regulation of proteolytic cascades.
Project description:Serpins (serine protease inhibitors) regulate some innate immune responses of insects by inhibiting endogenous proteases. In this study, we characterized the serpin (SRPN) gene family in the mosquito Anopheles gambiae, the major malaria vector in Sub-Saharan Africa. We identified 18 A. gambiae SRPN genes, all on chromosomes 2 and 3, through searches of genomic DNA and EST databases. In addition to SRPN10, previously documented to exhibit alternative splicing, we found three splicing isoforms of SRPN4. We completed sequencing of cDNAs for the A. gambiae serpins to obtain complete coding sequence information and to verify or improve gene predictions. The predicted SRPN9 and 15 in the initial genome annotation were determined to be a single gene (SRPN9). Sixteen of the serpins contained putative secretion signal sequences. Multiple sequence alignments showing conserved residues important in structural conformation, including the consensus pattern within the hinge region, indicated that most of the A. gambiae serpins may be inhibitory. Phylogenetic analyses confirmed that SRPN1, 2, 3, 8, 9 and 10 formed phylogenetic clusters with known inhibitory serpins from Drosophila melanogaster and Manduca sexta. Many of the A. gambiae serpins were expressed during all life stages. However, SRPN7, 8, 12, and 19 were expressed at very low levels in the adult stage. SRPN13 was expressed mostly in eggs and young larvae, whereas SRPN5 and 14 were expressed mostly in adults. Such differences in expression pattern suggest that the serpins are involved in multiple physiological processes. Determining the biological functions of the mosquito serpins will require future work to identify the proteases they inhibit in vivo.
Project description:Serpins form an enormous superfamily of 40-60-kDa proteins found in almost all types of organisms, including humans. Most are one-use suicide substrate serine and cysteine proteinase inhibitors that have evolved to finely regulate complex proteolytic pathways, such as blood coagulation, fibrinolysis, and inflammation. Despite distinct functions for each serpin, there is much redundancy in the primary specificity-determining residues. However, many serpins exploit additional exosites to generate the exquisite specificity that makes a given serpin effective only when certain other criteria, such as the presence of specific cofactors, are met. With a focus on human serpins, this minireview examines use of exosites by nine serpins in the initial complex-forming phase to modulate primary specificity in either binary serpin-proteinase complexes or ternary complexes that additionally employ a protein or other cofactor. A frequent theme is down-regulation of inhibitory activity unless the exosite(s) are engaged. In addition, the use of exosites by maspin and plasminogen activator inhibitor-1 to indirectly affect proteolytic processes is considered.
Project description:The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease.
Project description:Over the past 19 years, we have developed a novel myxoma virus-derived anti-inflammatory serine protease inhibitor, termed a serpin, as a new class of immunomodulatory therapeutic. This review will describe the initial identification of viral serpins with anti-inflammatory potential, beginning with preclinical analysis of viral pathogenesis and proceeding to cell and molecular target analyses, and successful clinical trial. The central aim of this review is to describe the development of two serpins, Serp-1 and Serp-2, as a new class of immune modulating drug, from inception to implementation. We begin with an overview of the approaches used for successful mining of the virus for potential serpin immunomodulators in viruses. We then provide a methodological overview of one inflammatory animal model used to test for serpin anti-inflammatory activity followed by methods used to identify cells in the inflammatory response system targeted by these serpins and molecular responses to serpin treatment. Finally, we provide an overview of our findings from a recent, successful clinical trial of the secreted myxomaviral serpin, Serp-1, in patients with unstable inflammatory coronary arterial disease.
Project description:Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and beta-1,3-glucan-mediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.
Project description:BACKGROUND: The serpin (serine protease inhibitor) superfamily constitutes a class of functionally highly diverse proteins usually encompassing several dozens of paralogs in mammals. Though phylogenetic classification of vertebrate serpins into six groups based on gene organisation is well established, the evolutionary roots beyond the fish/tetrapod split are unresolved. The aim of this study was to elucidate the phylogenetic relationships of serpins involved in surveying the secretory pathway routes against uncontrolled proteolytic activity. RESULTS: Here, rare genomic characters are used to show that orthologs of neuroserpin, a prominent representative of vertebrate group 3 serpin genes, exist in early diverging deuterostomes and probably also in cnidarians, indicating that the origin of a mammalian serpin can be traced back far in the history of eumetazoans. A C-terminal address code assigning association with secretory pathway organelles is present in all neuroserpin orthologs, suggesting that supervision of cellular export/import routes by antiproteolytic serpins is an ancient trait, though subtle functional and compartmental specialisations have developed during their evolution. The results also suggest that massive changes in the exon-intron organisation of serpin genes have occurred along the lineage leading to vertebrate neuroserpin, in contrast with the immediately adjacent PDCD10 gene that is linked to its neighbour at least since divergence of echinoderms. The intron distribution pattern of closely adjacent and co-regulated genes thus may experience quite different fates during evolution of metazoans. CONCLUSION: This study demonstrates that the analysis of microsynteny and other rare characters can provide insight into the intricate family history of metazoan serpins. Serpins with the capacity to defend the main cellular export/import routes against uncontrolled endogenous and/or foreign proteolytic activity represent an ancient trait in eukaryotes that has been maintained continuously in metazoans though subtle changes affecting function and subcellular location have evolved. It is shown that the intron distribution pattern of neuroserpin gene orthologs has undergone substantial rearrangements during metazoan evolution.