Under challenge by a dietary soybean cysteine protease inhibitor (scN), cowpea bruchids overcome the inhibitory effects by reconfiguring the expression profiles of their major digestive enzymes, the cathepsin L-like cysteine proteases (CmCPs). In addition, cowpea bruchids activate transcription of the counter-defensive cathepsin B-like cysteine protease (CmCatB). I undertook an interest in understanding the molecular mechanisms utilized by bruchids to differentially regulate cysteine proteases in response to plant inhibitors. First, to investigate the functional significance of the differential regulation of CmCPs, I expressed CmCP proprotein isoforms (proCmCPs) in E. coli, and characterized their activities. Among proCmCPs, proCmCPB1 exhibited the most efficient autocatalytic processing, the highest proteolytic activity, and was able to degrade scN in the presence of excessive CmCPB1. Second, to dissect the molecular mechanisms behind the differential function of CmCPs, I swapped domains between two representative subfamily members B1 and A16. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- or C-terminal mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16. Bacterially expressed isolated propeptides (pA16 and pB1) showed that pB1 inhibited B1 enzyme less than pA16 due to its protein instability. Taken together, these results suggest that cowpea bruchids selectively induce specific cysteine proteases for their superior autoprocessing, proteolytic efficacy, and scNdegrading activities, and modulate proteolysis of their digestive enzymes by controlling cleavage and stability of propeptides to cope with plant inhibitors. Third, to understand the transcriptional regulatory mechanisms of CmCatB hyperexpression that underlies bruchid adaptation, I cloned a portion of its promoter and demonstrated its activity in Drosophila S2 cells using a CAT reporter system. Gel shift assays identified cowpea bruchid Seven-up (CmSvp, chicken ovalbumin upstream promoter transcription factor homolog) in scN-unadapted insect midgut, and cowpea bruchid HNF-4 (CmHNF-4, hepatocyte nuclear factor 4) in scN-adapted insect midgut. When transiently expressed in S2 cells, CmSvp repressed, while CmHNF-4 activated CmCatB expression. CmSvp antagonized CmHNF-4-mediated transactivation when they were present simultaneously in the cell. Thus, the data suggest that transcriptional regulation of CmCatB in response to plant inhibitor depends, at least partly, on the cellular balance between positive and negative regulators.

A existência de uma família gênica diversa de serino proteinases em Lepidóptera sugere que essas proteinases desempenham um papel importante na adaptação desses insetos à presença de inibidores de proteinases vegetais. Essas enzimas têm se revelado estarem envolvidas no processo digestivo de larvas de insetos. Larvas de Spodoptera frugiperda foram alimentadas com uma dieta suplementada com inibidor de proteinase de soja (IPS) e a expressão gênica de proteinases intestinais foi avaliada através de PCR em tempo real. Análises de transcrição anteriores mostraram a existência de dois grupos de serino proteinases: um grupo de genes constitutivamente expressos em larvas controle que é induzido pela dieta contendo IPS e um segundo grupo que está ausente no controle, mas que é também induzido por uma dieta rica em IPS. No presente trabalho foi observado um terceiro grupo de proteinases que não são nem induzidas nem reprimidas pela presença do IPS na dieta. Essa observação sugere que a adaptação de S. frugiperda ao IPS envolve a síntese de novas proteinases, a indução de enzimas preexistentes e ainda um terceiro grupo insensível à presença dos inibidores. Proteinases dos intestinos de larvas crescidas em dieta com IPS mostraram insensibilidade ao inibidor. As proteinases também foram insensíveis quando a atividade foi verificada com um inibidor de proteinases de amplo espectrum. Os resultados aqui apresentados propõem que a adaptação de S. frugiperda ao IPS segue uma estratégia generalizada, baseada na indução geral de um grande grupo de endoproteinases. / The existence of a diverse serine proteinase gene family in lepidopteran insects has suggested its significant role in the insect adaptation to plant proteinase inhibitors. These enzymes have been shown to be involved in the proteolytic digestion process of insect larvae. Spodoptera frugiperda larvae were fed on a diet supplemented with soybean proteinase inhibitor (SPI) and the gene expression of intestinal proteinases was evaluated by real time PCR. Previous transcription analyses found two groups of intestinal serine proteinases: one group of genes constitutively expressed in the control larvae that is induced by the SPI-containing diet during the experiment, and a second group that is absent in the control but also induced by the SPI rich diet. Herein was observed a third group of proteinases that are neither induced nor repressed by the presence of SPI in the diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis, up regulation of existing enzymes and that there is a third group insensitive to the presence of the inhibitors. Proteinases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteinases were also insensitive when the activity was checked with a broad-spectrum potato proteinase inhibitor. The results here presented propose that adaptation of S. frugiperda to SPI follows a \"shotgun\" approach, based on a general up regulation of a large set of endoproteinases.

A existência de uma família gênica diversa de serino proteinases em Lepidóptera sugere que essas proteinases desempenham um papel importante na adaptação desses insetos à presença de inibidores de proteinases vegetais. Essas enzimas têm se revelado estarem envolvidas no processo digestivo de larvas de insetos. Larvas de Spodoptera frugiperda foram alimentadas com uma dieta suplementada com inibidor de proteinase de soja (IPS) e a expressão gênica de proteinases intestinais foi avaliada através de PCR em tempo real. Análises de transcrição anteriores mostraram a existência de dois grupos de serino proteinases: um grupo de genes constitutivamente expressos em larvas controle que é induzido pela dieta contendo IPS e um segundo grupo que está ausente no controle, mas que é também induzido por uma dieta rica em IPS. No presente trabalho foi observado um terceiro grupo de proteinases que não são nem induzidas nem reprimidas pela presença do IPS na dieta. Essa observação sugere que a adaptação de S. frugiperda ao IPS envolve a síntese de novas proteinases, a indução de enzimas preexistentes e ainda um terceiro grupo insensível à presença dos inibidores. Proteinases dos intestinos de larvas crescidas em dieta com IPS mostraram insensibilidade ao inibidor. As proteinases também foram insensíveis quando a atividade foi verificada com um inibidor de proteinases de amplo espectrum. Os resultados aqui apresentados propõem que a adaptação de S. frugiperda ao IPS segue uma estratégia generalizada, baseada na indução geral de um grande grupo de endoproteinases. / The existence of a diverse serine proteinase gene family in lepidopteran insects has suggested its significant role in the insect adaptation to plant proteinase inhibitors. These enzymes have been shown to be involved in the proteolytic digestion process of insect larvae. Spodoptera frugiperda larvae were fed on a diet supplemented with soybean proteinase inhibitor (SPI) and the gene expression of intestinal proteinases was evaluated by real time PCR. Previous transcription analyses found two groups of intestinal serine proteinases: one group of genes constitutively expressed in the control larvae that is induced by the SPI-containing diet during the experiment, and a second group that is absent in the control but also induced by the SPI rich diet. Herein was observed a third group of proteinases that are neither induced nor repressed by the presence of SPI in the diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis, up regulation of existing enzymes and that there is a third group insensitive to the presence of the inhibitors. Proteinases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteinases were also insensitive when the activity was checked with a broad-spectrum potato proteinase inhibitor. The results here presented propose that adaptation of S. frugiperda to SPI follows a \"shotgun\" approach, based on a general up regulation of a large set of endoproteinases.