In this study we describe the first cysteine proteinases of the MEROPS Clan CD family C13 in Fasciola gigantica. Family C13 contains asparaginyl endopeptidases and glycosylphosphatidylinositol-anchor transamidases and is also called the legumain family due to the discovery of the first asparaginyl endopeptidase in a legume. The cDNAs encoding two asparaginyl endopeptidases, FgLGMN-1 and FgLGMN-2, were cloned and used for the analysis of nucleic acid and protein properties. The deduced amino acid sequences showed 47.4% identity to each other and from 42.2 to 51.1% identity to homologs of other trematode species. The catalytic site residues histidine, cysteine and preceding hydrophobic residues, characteristic for the cysteine proteinase families C11, C13, C14, and C25, were found conserved. Northern and reverse transcription PCR analyses demonstrated that the transcriptional products are present in metacercariae, juveniles and adults. RNA in situ hybridization and immunohistochemistry revealed that RNA and protein products of the two genes are specifically expressed in the intestinal epithelium of juveniles and adults. Immune sera of mice infected with F. gigantica reacted with immunoblotted, bacterially expressed recombinant proteins starting 4 weeks after infection. Polyclonal antisera raised against the recombinant proteins detected 40 and 30 kDa antigens, respectively in crude worm protein extracts but not in the excretion-secretion products of adult parasites. Likewise, legumain-specific activity was found in crude worm protein extracts but not in excretion-secretion products. This study elucidates the molecular characteristics of these proteins in F. gigantica and demonstrates differences in the biology between Fasciola and Schistosoma which may prove useful for the development of vaccines against fasciolosis in domestic livestock.

Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.

Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-γ-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.

The metabolic pathways of protein during anaerobic digestion (AD) of waste activated sludge (WAS) were comprehensively investigated. Results showed that 100 kinds of peptidases were involved in the hydrolysis and acidogenesis processes. Serine endopeptidases (EC 3.4.21.53) and serine-type carboxypeptidases (EC 3.4.16.4) were the key enzymes of endopeptidases and exopeptidases, respectively. The pathways of ko00250 (alanine, aspartate and glutamate metabolism), ko00260 (glycine, serine and threonine metabolism), ko00270 (cysteine and methionine metabolism), ko00280 (valine, leucine and isoleucine degradation), ko00360 (phenylalanine metabolism) and ko00310 (lysine degradation) were the critical metabolic pathways of amino acids during AD of WAS, since they have complete pathways from amino acids to vital intermediates (pyruvate or acetyl-CoA). l-aspartate, l-alanine, threonine, glycine, serine, l-cysteine were the intermediate products in the conversion of protein to pyruvate, while l-leucine, l-isoleucine, phenylalanine, lysine could be directly metabolized to acetyl-CoA. Dechloromonas and Thauera played major roles in the crucial metabolic pathways of amino acids (ko00250, ko00260, ko00280 and ko00270). These important discoveries could provide a new biological perspective for improving AD performance.

Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases.

Marafiviruses are capable of persistent infection in a range of plants that have importance to the agriculture and biofuel industries. Although the genomes of a few of these viruses have been studied in-depth, the composition and processing of the polyproteins produced from their main ORFs have not. The Marafivirus polyprotein consists of essential proteins that form the viral replicase, as well as structural proteins for virus assembly. It has been proposed that Marafiviruses code for cysteine proteases within their polyproteins, which act as endopeptidases to autocatalytically cleave the polyprotein into functional domains. Furthermore, it has also been suggested that Marafivirus endopeptidases may have deubiquitinating activity, which has been shown to enhance viral replication by downregulating viral protein degradation by the ubiquitin (Ub) proteasomal pathway as well as tampering with cell signaling associated with innate antiviral responses in other positive-sense ssRNA viruses. Here, we provide the first evidence of cysteine proteases from six different Marafiviruses that harbor deubiquitinating activity and reveal intragenus differences toward Ub linkage types. We also examine the structural basis of the endopeptidase/deubiquitinase from the Marafivirus type member, maize rayado fino virus. Structures of the enzyme alone and bound to Ub reveal marked structural rearrangements that occur upon binding of Ub and provide insights into substrate specificity and differences that set it apart from other viral cysteine proteases.

Allosteric targeting is progressively gaining ground as a strategy in drug design. Its success, however, depends on our knowledge of the investigated system. In the case of the papain-like cysteine peptidase cathepsin K, a major obstacle in our understanding of allostery is represented by the lack of observable conformational change at the active site. This makes it difficult to understand how binding of effectors at known allosteric sites translates into modified enzyme activity. Herein, we address this issue by a computational approach based on experimental data. We analyze the conformational space of the papain-like family and the positioning of cathepsin K within it using principal component analysis and molecular dynamics simulations. We show that human cathepsin L-like endopeptidases (cathepsins L, K, S and V) adopt similar conformations which are distinct from their non-animal counterparts and other related peptidases. Molecular dynamics simulations show that the conformation of cathepsin K is influenced by known allosteric effectors, chondroitin sulfate and the small molecules NSC13345 and NSC94914. Importantly, all effectors affect the geometry of the active site around sites S1 and S2 that represent the narrowest part of the active site cleft and the major specificity determinant in papain-like endopeptidases. The effectors act by stabilizing pre-existing conformational states according to a two-state model and thereby facilitate or hinder the binding of substrate into the active site, as shown by molecular docking simulations. Comparison with other related enzymes shows that similar conformational variability and, by implication, allostery also exist in other papain-like endopeptidases.

Human α2-macroglobulin (hα2M) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hα2M undergoes stepwise transitions from its native, expanded, highly flexible, active conformation to an induced, compact, triggered conformation. As a consequence, the peptidase is entrapped by an irreversible Venus flytrap mechanism. Given the importance of hα2M, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hα2M purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hα2M is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hα2M. We therefore used a combination of native and sodium dodecylsulfate polyacrylamide gel electrophoresis, affinity and ion-exchange chromatography, multi-angle laser light scattering after size-exclusion chromatography, free cysteine quantification, and peptidase inhibition assays with endopeptidases of two catalytic classes and three protein substrates, to characterize the biochemical and biophysical properties of hα2M purified ad hoc either from fresh plasma or frozen fresh plasma after thawing. We found no differences in the molecular or functional properties of the preparations, indicating that protective components in plasma maintain native hα2M in a functionally competent state despite freezing.

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1.

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.

The establishment of plant-fungus mutualistic interaction requires bidirectional molecular crosstalk. Therefore, the analysis of the interacting organisms secretomes would help to understand how such relationships are established. Here, a gel-free shotgun proteomics approach was used to identify the secreted proteins of the plant Arabidopsis thaliana and the mutualistic fungus Trichoderma atroviride during their interaction. A total of 126 proteins of Arabidopsis and 1027 of T. atroviride were identified. Among them, 118 and 780 were differentially modulated, respectively. Bioinformatic analysis unveiled that both organisms' secretomes were enriched with enzymes. In T. atroviride, glycosidases, aspartic endopeptidases, and dehydrogenases increased in response to Arabidopsis. Additionally, amidases, protein-serine/threonine kinases, and hydro-lyases showed decreased levels. Furthermore, peroxidases, cysteine endopeptidases, and enzymes related to the catabolism of secondary metabolites increased in the plant secretome. In contrast, pathogenesis-related proteins and protease inhibitors decreased in response to the fungus. Notably, the glutamate:glyoxylate aminotransferase GGAT1 was secreted by Arabidopsis during its interaction with T. atroviride. Our study showed that GGAT1 is partially required for plant growth stimulation and on the induction of the plant systemic resistance by T. atroviride. Additionally, GGAT1 seems to participate in the negative regulation of the plant systemic resistance against B. cinerea through a mechanism involving H2O2 production.

Three triticale cDNAs encoding inhibitors of cysteine endopeptidases, belonging to phytocystatins, have been identified and designated as TrcC-1, TrcC-4 and TrcC-5. Full-length cDNAs of TrcC-1 (617 bp) and TrcC-4 (940 bp), as well as a fragment of TrcC-5 cDNA (369 bp), were obtained. A high-level identity of the deduced amino acid sequence of TrcCs with other known phytocystatins, especially with wheat and barley, has been observed. Moreover, the presence of conserved domain, containing the G and W residues, the sequence of QxVxG and the sequence of LARFAV, characteristic for plant cysteine endopeptidase inhibitors, has been noted. The profiles of TrcC-1 and TrcC-5 mRNA levels in the developing seeds of two triticale cultivars that differ in their resistance to preharvest sprouting (Zorro and Disco) were similar. However, the expression of TrcC-4 was, higher in the developing seeds, and in the scutellum of germinating seeds of a cultivar more resistant to preharvest sprouting (Zorro) than in the less resistant (Disco). Additionally, the expression of TrcC-4 remained longer in developing seeds of Zorro as compared to Disco. The performed studies suggest that TrcC-4 might have an influence on the higher resistance of Zorro cultivar to preharvest sprouting.

Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-D-glutamic acid and L-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure-activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1-Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.

Molecular genetic markers of various PCD (programmed cell death) variants during xylo- and phloemogenesis have been identified for the first time in Scots pine under lingonberry pine forest conditions in Northwest Russia (middle taiga subzone). PCD is a genetically determined process. Gene profiles of serine and cysteine proteases (endopeptidases), endonucleases, and metacaspases families are often considered markers of the final xylogenesis stage. In the present study, we examined the gene expression profiles of the BFN (bifunctional endonuclease) family-BFN, BFN1, BFN2, BFN3, and peptidase (cysteine endopeptidase, CEP and metacaspase, MC5) in the radial row, in addition to the vascular phloem and cambium (F1), differentiating xylem (F2), sapwood (SW), and transition zone during the active cambial growth period of uneven-aged pine trees (25-, 63- and 164-cambial age (c.a.) years old). We have shown that the expression patterns of the PCD-related genes did not depend on the cambial age but were largely determined by plant tissue type. In the radial row F1-F2-SW, we studied the activities of enzymes, including sucrose in metabolism (sucrose synthase, three forms of invertase); antioxidant system (AOS) enzymes (superoxide dismutase, catalase); and peroxidase andpolyphenol oxidase, which belonged to AOS enzymes and were involved in the synthesis of phenolic components of cell walls. The activity of the enzymes indicated that the trunk tissues of pine trees had varying metabolic status. Molecular genetic PCD regulation mechanisms during xylem vascular and mechanical element formation and parenchyma cells' PCD during the formation of Scots pine heartwood were discussed.

Serine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques.

Thelohanellus kitauei, is a member of obligate parasitic myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite's survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G19G20, Q73VVAG77, and C-terminal L102P103. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (- 13 KJ/mol) with cathepsin L whereas a higher binding energy (- 8.6 KJ/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-T. kitauei vaccine in carp culture in the future.

Yersinia ruckeri is the causative agent of enteric redmouth disease in salmonids. In fish, the intestine represents an important site of nutrient uptake, host-pathogen interactions, and defense. The posterior intestine can be inflamed, reddened, and filled with an opaque, yellowish fluid during Y. ruckeri infection. Herein, we report an investigation on the proteome alteration in the posterior intestinal mucosa of rainbow trout (Oncorhynchus mykiss) after exposure to Y. ruckeri. The intestinal mucosal proteins were identified and quantified by a shotgun proteomic approach by applying data-independent quantification with sequential windowed acquisition of all theoretical mass spectra (SWATH). A total of 437 proteins were found to be differentially up- or downregulated in the posterior intestine. Gene ontology of upregulated proteins pointed to their involvement into exopeptidase, endopeptidase, and hydrolase activities, while the downregulated proteins were involved in lipid metabolism, actin binding, and translation processes. Additionally, upregulated proteins were predicted to be involved in lysosome, oxidative phosphorylation, and metabolic pathways, while downregulated proteins were implicated in focal adhesion, regulation of actin cytoskeleton, protein digestion and absorption pathways. This study showed that Y. ruckeri infection can alter protein abundance involved in serine-type carboxypeptidase, cysteine and aspartic-type endopeptidases, metallopeptidases, antioxidant defense, calcium ion binding, glycolytic and carbohydrate metabolic processes in the proteome of the intestinal mucosa of rainbow trout.

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

Chitin-binding hevein-like peptides (CB-HLPs) belong to a family of cysteine-rich peptides that play important roles in plant stress and defense mechanisms. CB-HLPs are ribosomally synthesized peptides that are known to be bioprocessed from the following two types of three-domain CB-HLP precursor architectures: cargo-carrying and non-cargo-carrying. Here, we report the identification and characterization of chenotides biosynthesized from the third type of precursors, which are cleavable hololectins of the quinoa (Chenopodium quinoa) family. Chenotides are 6-Cys-CB-HLPs of 29-31 amino acids, which have a third type of precursor architecture that encompasses a canonical chitin-binding domain that is involved in chitin binding and anti-fungal activities. Microbroth dilution assays and microscopic analyses showed that chenotides are effective against phyto-pathogenic fungi in the micromolar range. Structure determination revealed that chenotides are cystine knotted and highly compact, which could confer resistance against heat and proteolytic degradation. Importantly, chenotides are connected by a novel 18-residue Gly/Ala-rich linker that is a target for bioprocessing by cathepsin-like endopeptidases. Taken together, our findings reveal that chenotides are a new family of CB-HLPs from quinoa that are synthesized as a single multi-modular unit and bioprocessed to yield individual mature CB-HLPs. Importantly, such precursors constitute a new family of cleavable hololectins. This unusual feature could increase the biosynthetic efficiency of anti-fungal CB-HLPs, to provide an evolutionary advantage for plant survival and reproduction.

Lepidopteran insect pest Helicoverpa armigera is one of the most destructive pests of crop plants and several biotechnological approaches are being developed for its control. Plant defensins are small cationic and cysteine-rich peptides that play a role in plant defense. Ingestion of a defensin from Capsicum annuum (CanDef-20) induced a dose-dependent reduction in larval and pupal mass, delayed metamorphosis and also severely reduced fecundity and fertility in H. armigera. To understand the molecular mechanisms of CanDef-20 ingestion-mediated antibiosis in H. armigera larvae, a comparative transcriptomics analysis was carried out. Predominant downregulation of GOs represents serine-type endopeptidases, structural constituents of ribosomes and integral membrane components and differential upregulation of ATP binding, nucleus and translation, while up-regulation of nucleic acid binding represented by transposable elements, were detected. Different isoforms of lipase, serine endopeptidase, glutathione S-transferase, cadherin, alkaline phosphatase and aminopeptidases were found to be upregulated as a compensatory response to CanDef-20 ingestion. In vitro enzyme assays and qPCR analysis of some representative genes associated with vital cellular processes like metamorphosis, food digestion and gut membrane indicated adaptive differential regulations in CanDef-20 fed H. armigera larvae. We conclude that CanDef-20 ingestion affects insect metabolism in a number of ways through its interaction with cell membrane, enzymes, cytoplasmic proteins and triggering transposon mobilization which are linked to growth retardation and adaptive strategies in H. armigera.