RESUMO
In insects the enzyme phenoloxidase (PO) catalyzes melanin deposition at the wound site and around parasitoid eggs. Its proenzyme prophenoloxidase (proPO) is proteolytically cleaved to active phenoloxidase by a cascade consisting of serine proteases and inhibited by serpins. The Drosophila genome encodes 29 serpins, of which only two, Serpin-27A (Spn27A) and Necrotic, have been analyzed in detail. Using a genetic approach, we demonstrate that the so far uncharacterized Serpin-28D (Spn28D, CG7219) regulates the proPO cascade in both hemolymph and tracheal compartments. spn28D is the serpin gene most strongly induced upon injury. Inactivation of spn28D causes pupal lethality and a deregulated developmental PO activation leading to extensive melanization of tissues in contact with air and pigmentation defects of the adult cuticle. Our data also show that Spn28D regulates hemolymph PO activity in both larvae and adults at a different level than Spn27A. Our data support a model in which Spn28D confines PO availability by controlling its initial release, while Spn27A is rather limiting the melanization reaction to the wound site. This study further highlights the complexity of the proPO cascade that can be differentially regulated in different tissues during development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Hemolinfa/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Pigmentação , Serpinas/metabolismo , Animais , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Larva/enzimologia , Melaninas/metabolismo , Mutação/genética , Fenótipo , Pupa/enzimologia , Interferência de RNA , Traqueia/anormalidadesRESUMO
Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.
Assuntos
Coagulação Sanguínea/fisiologia , Drosophila melanogaster/fisiologia , Hemolinfa/metabolismo , Animais , Fenômenos Fisiológicos Bacterianos , Western Blotting , Proteínas de Drosophila/sangue , Drosophila melanogaster/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/sangue , Lectinas/sangue , Espectrometria de Massas , Microesferas , Monofenol Mono-OxigenaseRESUMO
Our studies on the developmental regulation of glycosylation in Drosophila melanogaster led us to identify and characterize gp150, an ecdysone-regulated mucin that is found in hemocytes, the gut (peritrophic membrane) and in the salivary glands. We are particularly interested in mucin immune functions and found that gp150 is released from larval hemocytes, becomes part of the clot and participates in the entrapment of bacteria. By RT-PCR and RNAi experiments, we identified gp150 as the previously described I71-7, an ecdysone-induced salivary glue protein. We discuss the evolutionary and biochemical implications of the dual use of salivary proteins for immune functions in insects. Further molecular characterization of such shared proteins may enable a better understanding of the properties of proteins involved in containment and elimination of microbes, as well as hemostasis and wound repair.
Assuntos
Drosophila melanogaster/imunologia , Proteínas de Insetos/biossíntese , Proteínas de Insetos/fisiologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/fisiologia , Mucinas/biossíntese , Mucinas/fisiologia , Sequência de Aminoácidos , Animais , Bactérias/imunologia , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Ecdisona/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Glicosilação , Hemócitos , Hemolinfa , Imunidade Inata , Larva/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares/metabolismoRESUMO
Organismal lifespan has been the primary readout in aging research. However, how longevity genes control tissue-specific aging remains an open question. To examine the crosstalk between longevity programs and specific tissues during aging, biomarkers of organ-specific aging are urgently needed. Since the earliest signs of aging occur in the skin, we sought to examine skin aging in a genetically tractable model. Here we introduce a Drosophila model of skin aging. The epidermis undergoes a dramatic morphological deterioration with age that includes membrane and nuclear loss. These changes were decelerated in a long-lived mutant and accelerated in a short-lived mutant. An increase in autophagy markers correlated with epidermal aging. Finally, the epidermis of Atg7 mutants retained younger characteristics, suggesting that autophagy is a critical driver of epidermal aging. This is surprising given that autophagy is generally viewed as protective during aging. Since Atg7 mutants are short-lived, the deceleration of epidermal aging in this mutant suggests that in the epidermis healthspan can be uncoupled from longevity. Because the aging readout we introduce here has an early onset and is easily visualized, genetic dissection using our model should identify other novel mechanisms by which lifespan genes feed into tissue-specific aging.
Assuntos
Envelhecimento/fisiologia , Autofagia/fisiologia , Drosophila/fisiologia , Fenômenos Fisiológicos do Tegumento Comum , Animais , Proteína 7 Relacionada à Autofagia , Biomarcadores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/fisiologia , Mutação , Vertebrados/fisiologiaRESUMO
Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Hemolinfa/fisiologia , Receptores Toll-Like/metabolismo , Animais , Coagulação Sanguínea , Proteínas Sanguíneas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Larva , Pupa , Interferência de RNA , Transdução de Sinais , Receptores Toll-Like/imunologiaRESUMO
Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.