Molecular Mechanisms Governing the Differential Regulation of Cysteine Proteases in Insect Adaptation to a Soybean Protease Inhibitor

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.