| Summary: | 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.
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