Walter R. Terra

Peptidases constitute at least 2% of genes in living organisms and participate in nearly all physiological processes across life forms. Conversely, peptidase inhibitors are essential for regulating proteolytic activity and have been widely applied. Combining high-throughput sequencing of novel peptidase inhibitor sources with molecular modeling and drug design currently represents an efficient strategy for developing new molecules. Venomous spiders harbor a wide array of peptidase inhibitors in both their venom and digestive system. However, biochemical and transcriptomic investigations of non-venomous spiders (Uloboridae) remain recent and scarce. Here, transcriptomic and biochemical analyses of the Uloboridae spider Zosis geniculata’s digestive midgut diverticula (MD) revealed that this species exhibited a digestive enzyme profile similar to that of other spiders. Furthermore, the MD transcriptome identified 19 peptidase inhibitors belonging to six inhibitor families. Serine peptidase inhibitors were the most abundant and diverse, while metallopeptidases represented the main proteolytic enzymes, suggesting that these inhibitors may have evolved to counteract prey-derived peptidases. Inhibitory assays using trypsin from potential insect prey confirmed this activity. The diversity and abundance of these molecules highlight Uloboridae spiders as promising novel sources of proteolytic inhibitors.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N₅₀ of 53.6 Mb, and L₅₀ of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

Serine peptidases (SPs) are responsible for most primary protein digestion in Lepidoptera species. An expansion of the number of genes encoding trypsin and chymotrypsin enzymes and the ability to upregulate the expression of some of these genes in response to peptidase inhibitor (PI) ingestion have been associated with the adaptation of Noctuidae moths to herbivory. To investigate whether these gene family expansion events are common to other Lepidoptera groups, we searched for all genes encoding putative trypsin and chymotrypsin enzymes in 23 publicly available genomes from this taxon. Phylogenetic analysis showed that several gene family expansion events may have occurred in the taxon’s evolutionary history and that these events gave rise to a very diverse group of enzymes, including proteins lacking the canonical SP catalytic triad. The expression profile of these enzymes along the midgut and the secretion mechanisms by which these enzymes enter the luminal content were also analyzed in Spodoptera frugiperda larvae using RNA-seq and proteomics. These results support the proposal of a midgut countercurrent flux responsible for the direction of these proteins to the anterior portion of the midgut and show that these enzymes reach the midgut lumen via both exocytosis and microapocrine secretion mechanisms.

Aminopeptidase M (AMP) inhibition is of interest for several diseases, such as highly vascularized cancer types. AMP can be inhibited by linear pentapeptides isolated from Microcystis aeruginosa LTPNA08 (MG7XX). Porcine AMP inhibition—a model for human AMP—activity was spectrophotometrically measured by the formation of p-nitroanilide from L-leucine-p-nitroanilide substrate by AMP. AMP inhibition by MG770 exhibited comparable inhibition levels to amastatin (IC₅₀ values: 1.20 ± 0.1 μM and 0.98 ± 0.1 μM, respectively), while MG756 was slightly less potent (with IC₅₀ values of 3.26 ± 0.5 μM). Molecular modelling suggests a potential binding mode, based on the interaction with the Zn²⁺ cofactor, where MG770′s extra methyl group contributes to the disturbance of the Zn²⁺ cofactor complex and highlights the importance of hydrophobicity for the site.

A role of gelsolin in opening the way along the microvilli for secretory vesicles during microapocrine secretion is proposed here. Data obtained with different techniques showed that many digestive enzymes are released by microapocrine secretion in insects. Proteins that might be involved in the machinery of microapocrine secretion were selected from our transcriptomes and literature searches. The proteins were annexin, Complex actin‐related proteins 2 and 3 (ARP 2/3) cofilin, fimbrin, gelsolin 1, gelsolin 2, moesin, myosin 1, myosin 6, protein disulphide isomerase 1 (PDI 1), PDI 2 and profilin. The cDNAs coding for annexin, fimbrin, gelsolin 1, myosin 1, PDI 1 and PDI 2 were cloned and their sequences deposited in GenBank. Only gelsolin 1 and myosin 1 are expressed exclusively in the midgut (semiquantitative reverse transcriptase PCR). As myosin 1 may have a structural role in microvilli, gelsolin 1 is the best guess to be involved in the secretory machinery. A truncated recombinant gelsolin 1 was used to generate antibodies with which it was shown labelling inside and around midgut cell microvilli shown in an electron microscope, reinforcing a microvillar role for gelsolin 1. Suppression of gelsolin 1 synthesis by RNA interference prevents secretory vesicles from advancing inside the microvilli, in agreement with its putative role in severing the actin filaments to free the way for the vesicles.

We compared the whole complement of midgut carboxypeptidases from 10 insects pertaining to five orders based on transcriptomes obtained by deep sequencing and biochemical data. Most of the carboxypeptidases were metallocarboxypeptidases from family M14, with carboxypeptidase A (CPA) predominating over carboxypeptidase B (CPB). They were found in all of the insects studied except for the hemipterans and a bruchid beetle. M14 carboxypeptidases were expressed only in the midgut of Spodoptera frugiperda (Lepidoptera). The most expressed CPA from this insect (SfCPA) was cloned, sequenced and expressed as a recombinant enzyme. This enzyme was used to generate antibodies used to demonstrate that SfCPA is secreted by an exocytic route. Serine carboxypeptidases from family S10 were found in all of the insects studied here. In S. frugiperda, they are expressed in all tissues besides the midgut, in accordance with their presumed lysosomal role. In the hemipteran Dysdercus peruvianus, S10 carboxypeptidases are expressed only in midgut, suggesting that they are digestive enzymes. This was confirmed by enzyme assays of midgut contents. Furthermore, the substrate specificity of D. peruvianus S10 carboxypeptidases are predicted to be one CPC (preferring hydrophobic residues) and one CPD (preferring basic residues), thus able to hydrolyse the peptides formed by their digestive cathepsin D and cathepsin L, respectively. The role of S10 carboxypeptidases in bruchid beetles are suggested to be the same as in hemipterans.

Both soluble (SfTre1) and membrane‐bound (SfTre2) trehalases occur along the midgut of Spodoptera frugiperda larvae. Released SfTre2 was purified as a 67 kDa protein. Its K _m (1.6 mM) and thermal stability (half life 10 min at 62 °C) are different from the previously isolated soluble trehalase (K _m = 0.47 mM; 100% stable at 62 °C). Two cDNAs coding for S. frugiperda trehalases have been cloned using primers based on consensus sequences of trehalases and having as templates a cDNA library prepared from total polyA‐containing RNA extracted from midguts. One cDNA codes for a trehalase that has a predicted transmembrane sequence and was defined as SfTre2. The other, after being cloned and expressed, results in a recombinant trehalase with a K _m value and thermal stability like those of native soluble trehalase. This enzyme was defined as SfTre1 and, after it was used to generate antibodies, it was immunolocalized at the secretory vesicles and at the glycocalyx of columnar cells. Escherichia coli trehalase 3D structure and sequence alignment with SfTre1 support a proposal regarding the residue modulating the pKa value of the proton donor.