*2.3. Ureases and Derived Peptides as Sources of Insecticidal and Fungitoxic (Poly)Peptides*

The insecticidal [50] and fungitoxic [51] effects of canatoxin were described before its characterization as an isoform of jack bean urease (JBU) [43]. In the first study of canatoxin's insecticidal effect, it became clear that only insects relying on cathepsin-like digestive enzymes (such as the cowpea weevil *Callosobruchus maculatus* and the kissing bug *Rhodnius prolixus*) are sensitive to the toxin, while insects with digestion based on trypsin-like enzymes (such as the tobacco hornworn *Manduca sexta* or the fruitfly *Drosophila melanogaster*) show no susceptibility. The hypothesis of a proteolytic activation of the toxin was then proposed [50]. The hydrolysis of canatoxin with *C. maculatus* digestive enzymes yielded a 10 kDa entomotoxic peptide named pepcanatox [52]. Our group has demonstrated through inhibition of cathepsin-like enzymes, that the enzymatic activity of cathepsin B (cysteine proteinase) and cathepsin D (aspartic proteinases) is necessary for the release of toxic fragments of canatoxin [50,52]. Cathepsin B is a cysteine proteinase that can act as an exopeptidase or endopeptidase at acidic pH [53]. Cleavage by cathepsin B has a preference for basic and hydrophobic amino acids [54]. Meanwhile, cathepsin D cleavage occurs at acidic pH and has a preference for hydrophobic residues [55]. Subsequently, the major proteolytic activities of midgut homogenates of *D. peruvianus* nymphs were shown in vitro to catalyze the release of pepcanatox from JBU [56]. Cysteine, aspartic and metalloproteinases are present in both homogenates. Fluorogenic substrates

containing JBU partial sequences flanking the N-terminal or the C-terminal portion of the entomotoxic peptide were efficiently cleaved by the *D. peruvianus* nymph midgut homogenates. Different classes of enzymes in the homogenates cleaved both substrates suggesting that in vivo the release of the entomotoxic peptide results from the concerted action of at least two different proteinases [56].

Jaburetox-2Ec, a recombinant peptide with 93 amino acids (~11 kDa) equivalent to pepcanatox, was produced heterologously in *Escherichia coli* from the corresponding sequence of the JBU isoform JBURE-II [57]. Here the term "peptide" is used solely to emphasize the fact that it is a fragment of a much larger protein regardless of its molecular mass. Later on, the peptide called simply Jaburetox was developed, with the same urease-derived sequence and the 6 His tail found in Jaburetox-2Ec, but lacking the V5 epitope present in the latter [58]. Both peptides, Jaburetox-2Ec and Jaburetox, display equivalent insecticidal activity, evidencing that the epitope V5 is not implied in their entomotoxicity [58].

Since one of the most well studied mechanisms of defense against insect pest is digestive enzyme inhibition, this possibility was explored by our group for ureases and derived peptides. As described by Carlini et al. [50] and Ferreira da-Silva et al. [52], canatoxin showed no inhibitory effect on the proteolytic (cathepsin B or D-like) or α-amylase activities. Moreover, the peptides derived from Canatoxin's digestion with cathepsin-like enzymes, including pepcanatox, did not display either proteolytic or amylase inhibitory properties [59]. Although Jaburetox itself was not tested, taking into account its virtually identical sequence when compared to pepcanatox, it is safe to assume that Jaburetox has no inhibitory effects upon digestive enzymes.

Jaburetox is lethal to several insects susceptible to canatoxin (the cotton stainer bug *D. peruvianus*, the kissing bugs *R. prolixus* and *Triatoma infestans*) and also kills insects that are resistant to intact ureases, such as lepidopterans (fall armyworm *Spodoptora frugiperda*, cotton bollworm *Helicoverpa armigera*) and dipterans (*Aedes aegypti*) [57,60], because the hydrolysis of the protein to release the peptide is no longer required.

Concerning the antifungal property of ureases and derived peptides, the most abundant jack bean isoform, JBU, was shown to display antifungal properties against a panel of 16 phytopathogenic filamentous fungi species of 11 genera, blocking spore germination and/or mycelial growth, and inhibiting multiplication of yeasts at submicromolar concentrations [47,61]. Jaburetox also displayed antifungal properties against filamentous fungi and yeasts [61].

Antifungal effects were observed in vitro also for isoforms of soybean urease (SBU) [47,62]. The participation of ureases in plant defense against fungal diseases was demonstrated in urease-null soybean plants obtained by gene silencing [63]. Later, the peptide called Soyuretox, homologous to Jaburetox, but derived from the ubiquitous isoform of the soybean urease, was heterologously expressed in *E. coli*, characterized structurally and its entomotoxic and antifungal effects were demonstrated [64].

Jaburetox showed no acute toxicity to mice and rats [57] and was found not toxic in a risk assessment study [65] while Soyuretox was not toxic to zebrafish embryos [64]. These data suggest that these peptides may be safe alternatives to attain resistance to insect herbivory and/or fungal disease in transgenic plants. In the following sections, the structural aspects (Figure 1) and biological profile of Jaburetox and Soyuretox are reviewed.
