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doi: 10.1242/10.1242/dev.00350

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Drosophila necrotic mutations mirror disease-associated variants of human serpins

Clare Green¹, Gemma Brown^1,*, Timothy R. Dafforn^2,, Jean-Marc Reichhart³, Terri Morley¹, David A. Lomas⁴ and David Gubb^1,

¹ Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK
² Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK
³ UPR 9022 CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France
⁴ Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, UK
^* Present address: Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
Present address: Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK

View larger version (85K): [in a new window]   Fig. 1. Dorsal thoracic region of nec null mutant fly (nec2/nec19). Within a few hours of eclosion, adult flies develop black cuticular patches that are associated with cellular necrosis of the underlying epithelial cells (Green et al., 2000). Necrotic patches are randomly distributed over most of the body surface, but occur preferentially at the proximal leg joints (arrows). Distal leg segments have been dissected away.

View larger version (42K): [in a new window]   Fig. 2. Models of serpin structure. (A) Position of nec mutations mapped onto the scaffold of monomeric 1-antitrypsin (Elliott et al., 2000). The GluLys substitution found in Z-1-antitrypsin, nec9 and nec20, maps at the hinge region between the reactive center loop (red) and ß-sheet A (green). ß-sheet B is colored yellow and -helix A is blue. NecS>F carries the Ser131Phe substitution homologous to that of 1-antitrypsin-Siiyama, Ser53Phe. (B) Loop-sheet polymer of Z-1-antitrypsin and Nec9.

View larger version (69K): [in a new window]   Fig. 3. Conformational stability of mutant serpins. (A) 7.5-15.0% (w/v) non-denaturing PAGE of cell extracts from flies carrying the E421K (nec9) mutation show reduced levels of native-like protein, N, and an increase in levels of higher molecular mass species, H. The more extreme phenotype of nec1 is associated with a further reduction in native protein. (B) 7.5% (w/v) TUG PAGE demonstrates that the higher molecular mass species (arrows) observed in both nec9 and nec1 are insensitive to denaturation in 8M urea. This behavior is characteristic of loop-sheet polymers such as those observed in the livers of individuals with Z-1-antitrypsin deficiency. These stabilized polymers are not observed in the wild-type flies. (C) The profile of monomeric 1-antitrypsin and polymerized Z-1-antitrypsin are shown for comparison, using purified proteins stained with Coomassie. The left of each gel represents 0 M urea and the right 8 M urea.

View larger version (27K): [in a new window]   Fig. 4. (A-C) Viability of nec9, nec1 and wild-type (WT) flies at 18°C (A), 25°C (B) and 29°C (C). The survival of nec9 flies, homologous to Z-variant 1-antitrypsin, is greatly reduced at 25°C and 29°C compared with that of wild-type flies. nec1 flies have shorter survival times than nec9, but survive about twice as long as complete null alleles (nec2/nec19, data not shown). (D-F) Viability of transgenic flies overexpressing wild-type or mutant Nec in a nec+ genetic background (Gal4-Act5c/+; necUAS/+) at 18°C (D), 25°C (E) and 29°C (F). (G) Viability of transgenic females overexpressing mutant Nec at 29°C in the fat body (Gal4-Yp/+; necUAS/+). Sibling males for these genotypes, in which the Yp promotor is inactive, were all healthy and the combined data for all three strains has been plotted as a control.