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Van Hoef V.,Zoological Institute Ku Leuven | Breugelmans B.,Zoological Institute Ku Leuven | Spit J.,Zoological Institute Ku Leuven | Simonet G.,Zoological Institute Ku Leuven | And 3 more authors.
Insect Biochemistry and Molecular Biology | Year: 2011

Introduction: In mammalian pancreatic cells, the pancreatic secretory trypsin inhibitor (PSTI) prevents the premature activation of digestive enzymes and thus plays an important role in a protective mechanism against tissue destruction by autophagy, a process which may ultimately cause diseases such as pancreatitis and pancreatic cancer. Insects, however, lack a pancreas and so far no PSTI-like peptides are functionally characterized. Results: In several insect species protease inhibitors that structurally resemble the mammalian PSTI were predicted in silico. A putative PSTI-like protein (LmPSTI) was cloned and sequenced in the African migratory locust, Locusta migratoria. For the first time the expression of an insect derived PSTI-like inhibitor was shown to be restricted to the digestive enzyme-producing organs in insects (midgut and caeca). LmPSTI was produced via a bacterial expression system and was found to be a potent inhibitor of bovine trypsin as well as endogenous locust gut enzymes. In the caeca, RNAi-mediated knockdown of LmPSTI resulted in a significantly upregulated expression (2-fold) of locust ATG8 transcripts (an ubiquitin-like protein crucial for autophagosome formation). These findings were confirmed by an ultrastructural study on caeca, revealing the presence of autophagy-related structures in RNAi-treated animals. Conclusion: The results of this study lead us to believe that LmPSTI plays an important role in controlling the proteolytic activity in the digestive system of L. migratoria. These findings provide new evidence for the existence of an ancient protective mechanism in metazoan digestive systems and open new perspectives for the study of autophagy-related diseases in the digestive tract. © 2011 Elsevier Ltd.

Spit J.,Zoological Institute Ku Leuven | Breugelmans B.,Zoological Institute Ku Leuven | Van Hoef V.,Zoological Institute Ku Leuven | Simonet G.,Zoological Institute Ku Leuven | And 2 more authors.
Peptides | Year: 2012

The main reason for the varying degrees of success of peptidase inhibitors (PI) as biological insecticides is the existence of a poorly understood mechanism, which allows pest insects to compensate for PI present in their diet. To challenge this highly flexible physiological mechanism and to prolong the inhibitory effect of PI on insect growth, a number of measures were taken into account before and during experiments with a notorious pest insect, the desert locust, Schistocerca gregaria: (i) non-plant PI (pacifastin-related inhibitors) were used to reduce the risk of a specific co-evolutionary adaptation of the pest insect, (ii) based on the main types of digestive enzymes present in the midgut, mixtures of multiple PI with different enzyme specificity were selected, allowing for a maximal inhibition of the proteolytic activity and (iii) digestive peptidase samples were taken during oral administration experiments to study compensatory mechanisms. Contrary to larvae fed on a diet containing plant-derived PI, a significant growth impediment was observed in larvae that were fed a mixture of different pacifastin-like PI. Nevertheless, the growth inhibition effect of this PI mixture attenuated after a few days, Moreover, a comprehensive study of the observed responses after oral administration of PI revealed that S. gregaria larvae can adjust their secreted digestive enzyme activities in two distinct ways depending on the composition/concentration of the PI-mixture. © 2011 Elsevier Inc. All rights reserved.

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