Carboxypeptidases and trehalases of insects

• Grant number: 12/07140-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2012
• End date: July 31, 2012
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Clelia Ferreira Terra
• Grantee: Katia Graziella de Oliveira Rebola
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 11/51685-8 - Insect digestion: a molecular, cellular, physiological and evolutionary approach, AP.TEM

Abstract

Digestion in insects: a molecular approach, cellular, physiological and evolutionary. Upon realization that the digestive tract is a relatively large interface with little protection between the insect and the environment, an understanding of intestinal function was considered essential to the development of new methods of control acting through the gut, such as the use of transgenic plants to control phytophagous insects. (A. Rev. Biochem. 61:87-111, 1992). Our laboratory chose to study in detail the strategy for the insect model arranged in the relevant points of the phylogenetic tree, to produce generalizations (A. Rev. Ent. 35: 181-200, 1990, Comp. Biochem. Physiol. B 109: 1 -62, 1994; Comprehensive Insect Molecular Science, vol. 4, Oxford, Elsevier, p. 171-224, 2005). The results showed that the digestive process in insects is organized phylogenetically certain standards and allowed raised hypotheses about the role of the peritrophic membrane (Arch. Insect Biochem. Physiol 47: 47-61, 2001) and membranes perimicrovillar of Hemiptera ( bugs, cicadas, and aphids) (An. Acad. Bras. Cienc. 78: 255-269, 2006). The development of the sector is the expansion of knowledge of the molecular events underlying the physiological phenomena bowel. This project addresses all aspects within the subdivision: (a) Study of digestive enzymes, (b) Molecular Physiology intestinal (c) Molecular basis of different mechanisms of intestinal secretion of enzymes, and (d) Evolution of digestive systems of insects. Digestive enzymes will be studied using procedures described in our review (already mentioned and more Insect Biochem. Molec. Biol. 35: 883-901, 2005, Acta Crys. F62: 750-752, 2006). The objective is to seek the causes of structural and kinetic peculiarities of the digestive enzymes that enable the insects to circumvent the defenses of plants. The following enzymes have their encoding cDNAs cloned, expressed, and subjected to recombinant proteins functional studies (site-directed mutation) and cristolográficos studies: trypsin and chymotrypsin P. American T. molitor, M. domestica and D. saccharalis, cathepsin L (CAL), and aminopeptidase N trehalase T. molitor, trehalase, b-glucosidase and laminarinase S. frugiperda, laminarinase flavolineata of th. The enzymes are then sequenced and characterized kinetically: CALs D. peruvianus, a-mannosidase T. molitor amylase and D. saccharalis. Models of the molecular physiology of intestinal D. peruvianus, T. molitor, M. domestica and S. frugiperda will be formulated in two steps: (a) random sequencing of cDNA libraries intestinal generation of antibodies for the most interesting clones, and their use in imunocitolocalizações to generate provisional hypotheses. (B) functional testing of hypotheses by suppressing the expression of proteins selected by RNAi and evaluation of the effects. The mechanisms of secretion of some enzymes in insects appear to include aspects that are not found in other animals (review). For these studies, scans of cDNA libraries intestinal expression with antibodies against cytoskeletal proteins microvilar (S. frugiperda), the apex cell (T. molitor) and microvillar (both insects) identify positive clones. These hypotheses bear secretion mechanism in two steps procedure, such as those mentioned for the study of the physiology molecular intestine. The development of the evolutionary hypothesis of the digestive systems of insects should include studies of digestive physiology of insects is not considered so far as cricket and walking stick and advance the study of membranes in perimicrovillar Thysanoptera.(AU)