Bacillus thuringiensis Crystal (Cry) and Cytolitic (Cyt) protein families are a diverse group of proteins with activity against insects of different orders--Lepidoptera, Coleoptera, Diptera and also against other invertebrates such as nematodes. Their primary action is to lyse midgut epithelial cells by inserting into the target membrane and forming pores. Among this group of proteins, members of the 3-Domain Cry family are used worldwide for insect control, and their mode of action has been characterized in some detail. Phylogenetic analyses established that the diversity of the 3-Domain Cry family evolved by the independent evolution of the three domains and by swapping of domain III among toxins. Like other pore-forming toxins (PFT) that affect mammals, Cry toxins interact with specific receptors located on the host cell surface and are activated by host proteases following receptor binding resulting in the formation of a pre-pore oligomeric structure that is insertion competent. In contrast, Cyt toxins directly interact with membrane lipids and insert into the membrane. Recent evidence suggests that Cyt synergize or overcome resistance to mosquitocidal-Cry proteins by functioning as a Cry-membrane bound receptor. In this review we summarize recent findings on the mode of action of Cry and Cyt toxins, and compare them to the mode of action of other bacterial PFT. Also, we discuss their use in the control of agricultural insect pests and insect vectors of human diseases.

Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture.

Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.

We previously reported that laminar flow activates peroxisome proliferator-activated receptor ␥ (PPAR␥) in vascular endothelial cells in a ligand-dependent manner that involves phospholipase A2 and cytochrome P450 epoxygenases. In this study, we investigated whether epoxyeicosatrienoic acids (EETs), the catalytic products of cytochrome P450 epoxygenases, are PPAR␥ ligands. Competition and direct binding assays revealed that EETs bind to the ligandbinding domain of PPAR␥ with Kd in the M range. In the presence of adamantyl-ureido-dodecanoic acid (AUDA), a soluble epoxide hydrolase (sEH)-specific inhibitor, EETs increased PPAR␥ transcription activity in endothelial cells and 3T3-L1 preadipocytes. Inclusion of AUDA in the perfusing media enhanced, but overexpression of sEH reduced, the laminar flow-induced PPAR␥ activity. Furthermore, laminar flow augmented cellular levels of EETs but decreased sEH at the levels of mRNA, protein, and activity. Blocking PPAR␥ by GW9662 abolished the EET͞AUDA-mediated antiinflammatory effect, which indicates that PPAR␥ is an effector of EETs. endothelial cells ͉ shear stressA therosclerosis preferentially localizes in branches and curved regions of the arterial tree, where the blood flow is disturbed. In contrast, the straight parts of vessels exposed to nondisturbed laminar flow have few lesions (1). The focal distribution of atherosclerotic lesions has been proposed to be related to the proinflammatory effect of disturbed flow imposed on the endothelium vs. the antiinflammatory effect of laminar flow. In vitro studies using flow channels with cultured endothelial cells (ECs) revealed that disturbed flow induces a number of molecules involved in inflammation, including chemoattractants, adhesion molecules, and cytokines (2, 3). However, prolonged exposure to laminar flow suppresses the cytokine-stimulated or oxidized low-density lipoprotein (LDL)-stimulated inflammatory response in ECs (4).Recent studies showed that the nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) is involved in antiinflammatory effects in the artery wall (5, 6). The activation of PPAR␥ in cultured ECs suppresses the NF-B-mediated expression of molecules such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and endothelin 1 that are involved in the inflammatory response (7,8). Troglitazone, a synthetic PPAR␥ ligand, attenuates the formation of lesions in both apolipoprotein E-and low-density lipoprotein receptordeficient mice (7, 9), due in part to the reduction of monocytes͞ macrophages homing to the plaques.We previously demonstrated that laminar flow activates PPAR␥ in a ligand-dependent manner, which exerts an antiinflammatory effect in ECs. Furthermore, we showed that such induction of PPAR␥ ligands involves phospholipase A2 and cytochrome P450 epoxygenases (CYPs) (10). Epoxyeicosatrienoic acids (EETs), the main products of arachidonic acid catalyzed by CYPs, have been reported to dilate coronary arteries by hyperpolarizing vascular smooth muscles (11) and to exe...