An Immune-Responsive Serpin Regulates the Melanization Cascade in Drosophila

Gregorio, Ennio De; Han, Sung‐Jun; Lee, Won Jae; Baek, Min Ji; Osaki, Tsukasa; Kawabata, Shun Ichiro; Lee, Bok Leul; Iwanaga, Sadaaki; Lemaître, Bruno; Brey, Paul T.

doi:10.1016/s1534-5807(02)00267-8

Cited by 309 publications

(275 citation statements)

References 36 publications

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“…De novo synthesis of melanin in response to tissue damage occurs through the melanization cascade, a series of enzymatic reactions involving serine proteases that activate phenol oxidase (PO), which catalyzes the conversion of phenolic substrates to quinones, which then polymerize to form melanin (32). The melanization reaction in the hemolymph can be blocked by mutations in the Drosophila gene Black cells (Bc), which encode PO, and Bc/Bc larvae do not have PO activity (33). To determine if the black spots in the Drosophila eyes expressing CHMP2B Intron5 are melanin deposits, we crossed GMR-Gal4, UAS-CHMP2B Intron5 flies with Bc mutant flies.…”

Section: Gmr-gal4; Uas-chmp2bmentioning

confidence: 99%

Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia

Ahmad

Sweeney

Lee

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

104

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Frontotemporal dementia (FTD) is the most common form of dementia before 60 years of age. Rare pathogenic mutations in CHMP2B, which encodes a component of the endosomal sorting complex required for transport (ESCRT-III), are associated with FTD linked to chromosome 3 (FTD3). Animal models of FTD3 have not yet been reported, and what signaling pathways are misregulated by mutant CHMP2B in vivo is unknown. Here we report the establishment of a Drosophila model of FTD3 and show the genetic interactions between mutant CHMP2B and other components of ESCRT. Through an unbiased genome-wide screen, we identified 29 modifier loci and found that serpin5 (Spn5), a largely uncharacterized serine protease inhibitor, suppresses the melanization phenotype induced by mutant CHMP2B in the fly eye. We also found that Spn5 is a negative regulator of the Toll pathway and functions extracellularly, likely by blocking the proteolytic activation of Spaetzle, the Toll receptor ligand. Moreover, Spn5 inhibited activation of the Toll pathway by mutant CHMP2B. Our findings identify Spn5 as a regulator of the Toll pathway and CHMP2B toxicity and show that the Toll pathway is a major signaling pathway misregulated by mutant CHMP2B in vivo. This fly model will be useful to further dissect genetic pathways that are potentially relevant to the pathogenesis and treatment of FTD.Drosophila ͉ endosomal sorting complex required for transport (ESCRT) ͉ neurodegeneration ͉ modifier screen F rontotemporal dementia (FTD), a major clinical syndrome of frontotemporal lobar degeneration (FTLD), is a progressive neurodegenerative condition associated with focal atrophy of the frontal and/or temporal lobes (1, 2). Although FTD is the most common form of senile dementia in people under 60 years of age, the molecular pathogenesis remains poorly understood (3). In some FTD brains, tau neurofibrillary tangles are present in diseased neurons, and some tau mutations are indeed pathogenic (4, 5). Several new genes have been implicated in FTD with tau-negative pathology, including those encoding valosin-containing protein (VCP) (6), CHMP2B (7), progranulin (8, 9), and TDP-43 (10, 11). The molecular pathways affected by these mutations and how they contribute to disease progression remain unclear.Although dominantly inherited CHMP2B mutations associated with FTD linked to chromosome 3 (FTD3) are rare (7,12,13), studies of CHMP2B neurotoxicity in cell culture models have been informative. CHMP2B is the ortholog of the yeast protein Vps2, a component of the endosomal sorting complex required for transport (ESCRT-III), which is involved in the biogenesis of multivesicular bodies and other biological processes (14). In undifferentiated PC12 cells, ectopic overexpression of CHMP2B Intron5 , a mutant form of CHMP2B missing 35 aa at the C terminus, led to the accumulation of vesicular structures (7). In cultured rodent cortical neurons and other cell types, CHMP2B Intron5 caused dendritic retraction, autophagosome accumulation, and neuronal cell loss (15,16). At the mol...

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Section: Gmr-gal4; Uas-chmp2bmentioning

confidence: 99%

Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia

Ahmad

Sweeney

Lee

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

104

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“…The soluble proteins of plasma, hemocyte lysate, and fat body were precipitated with trichloroacetic acid and subjected to SDS-PAGE and then immunoblotting with affinity-purified Hd-PGRP-1 and Hd-PGRP-2 antibodies, respectively. Previously it was reported that insect proPO system induced the activation of serine protease zymogen to active serine protease during 1,3-␤-D-glucan-dependent proPO activation (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)30). To explore the existence of serine protease activity during 1,3-␤-D-glucan-specific proPO activation, we examined the amidase activity in the presence of 1,3-␤-D-glucan and Ca 2ϩ by using commercially available trypsin substrate, Boc-PheSer-Arg-MCA.…”

Section: 3-␤-d-glucan and Pgn Binding Assay-mentioning

confidence: 99%

“…This enzyme produces toxic compounds to microorganisms by oxidizing phenols to melanin, and it also participates in the sclerotization of the cuticle, which is vital for the survival of insects (18). Many reports have been published about the proPO of the invertebrate and its activation mechanism (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). A key question regarding the proPO activation system is how pattern recognition molecules can induce activation of the system in response to microbial infection.…”

mentioning

confidence: 99%

Peptidoglycan Recognition Proteins Involved in 1,3-β-D-Glucan-dependent Prophenoloxidase Activation System of Insect

Lee

Osaki

Lee

et al. 2004

Journal of Biological Chemistry

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“…1) (Ashida and Brey, 1997;Sugumaran, 2002;Nappi and Christensen, 2005). In vitro and in vivo studies demonstrated that PO generates quinones for melanogenesis (Jiang et al, 1997;De Gregorio et al, 2002a) and leads to changes in the physical properties of hemolymph clot through protein crosslinking (Bidla et al, 2005). Cuticle tanning (i.e.…”

Section: Introductionmentioning

confidence: 99%

Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase

Zhao

Wang

et al. 2007

Insect Biochemistry and Molecular Biology

166

109

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Although quinone production and melanin formation are widely recognized as an integral part of the insect defense system, experimental evidence is lacking that the proteolytic activation of prophenoloxidase participates in the direct killing of invading microbes-active phenoloxidase generates quinones that polymerize to form melanin. Here, we report the antimicrobial effect of reactive intermediates produced in phenoloxidase-catalyzed reactions. After being treated with Manduca sexta phenoloxidase and dopamine, Escherichia coli and Bacillus subtilis ceased to grow, whereas the growth of Pichia pastoris was slightly affected. Microscopic analysis showed melanin deposition on cell surface, aggregation of bacteria, and loss of cell mobility. Viability tests revealed major decreases in the bacterial colony counts and, since the decrease remained significant after dispersion of the cell clumps, the reactive compounds were surmised to have aggregated and killed E. coli and B. subtilis cells. Under the experimental conditions, 60-94% of the Gram-negative bacteria (E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium) and 52-99% of the Gram-positive bacteria (Bacillus cereus, B. subtilis, Micrococcus luteus, and Staphylococcus aureus) were killed. In the presence of phenoloxidase, dopamine or 5,6-dihydroxyindole (DHI) exhibited much higher antibacterial activity than L-dopa, N-acetyldopamine (NADA) or N-β-alanyldopamine (NBAD) did, suggesting that DHI and its oxidation products were cytotoxic. The antifungal activity of DHI was detected using P. pastoris, Saccharomyces cerevisiae, Candida albicans, and Beauveria bassiana. These results established that prophenoloxidase activation is an integral component of the insect defense system involving a multitude of enzymes (e.g. proteinases, oxidases, and dopachrome conversion enzyme (DCE)), which immobilizes and kills invading microorganisms.

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An Immune-Responsive Serpin Regulates the Melanization Cascade in Drosophila

Cited by 309 publications

References 36 publications

Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia

Genetic screen identifies serpin5 as a regulator of the toll pathway and CHMP2B toxicity associated with frontotemporal dementia

Peptidoglycan Recognition Proteins Involved in 1,3-β-D-Glucan-dependent Prophenoloxidase Activation System of Insect

Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase

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