Rationale
Host defense peptides accumulated in the skin glands of the animals constitute the basis of the adaptive and immune system of amphibians. The peptidome of the Cuban frog Osteopilus septentrionalis was established using tandem mass spectrometry as the best analytical tool to elucidate the sequence of these peptides.
Methods
Manual interpretation of complementary collision‐induced dissociation (CID), higher energy collision‐induced dissociation (HCD), and electron transfer dissociation (ETD) tandem mass spectra recorded with an Orbitrap Elite mass spectrometer in liquid chromatography/mass spectrometry (LC/MS) mode was used to sequence the peptide components of the frog skin secretion, obtained by mild electrostimulation.
Results
Although the vast majority of amphibian peptides discovered so far are cationic, surprisingly only anionic peptides were identified in the skin secretion of the Cuban frog Osteopilus septentrionalis. Mass spectrometry allowed the sequences to be established of 16 representatives of new peptide families: septenins 1 and septenins 2. The highest sequence coverage when dealing with these anionic peptides was obtained with CID normalized collision energy 35 and HCD normalized collision energy 28.
Conclusions
Mirror‐symmetrical peptides are sequenced using N‐terminal acetylation. Acetylated Ser is reliably distinguished from isomeric Glu by the loss of ketene from b‐ions containing the corresponding residue. Calculations of the physicochemical and structural properties of the discovered anionic septenins 1 and 2 allowed the mechanism of their interaction with microbe cells to be postulated.
Aminopeptidases selectively hydrolyze an aminoacid residue from the amino terminus of proteins and peptides resulting in their activation or inactivation. These enzymes are mainly metallo and belong, among other, to the M1 family of peptidases. One of its members, membrane glutamyl aminopeptidase (APA, EC 3.4.11.7) participates in many physiological processes, such as peptide metabolism related with blood pressure control, and last step of protein degradation. Furthermore, the up regulation of APA has been implicated in the pathogenesis of various human disorders like cancers, hypertension and glomerulosclerosis. APA is thus a target for the development of inhibitors with potential biomedical applications. We review the most important structural and functional characteristics of mammalian APA, focusing on the most recent data. Additionally, we integrate the roles of APA in physio- and pathophysio-logical processes of biomedical relevance with the development of specific APA inhibitors.
Bufadienolides are steroids that inhibit the Na+/K+ ATPase pump. Recent studies show that members of the bufadienolide family also inhibit the activity of aminopeptidase N (APN). APN is upregulated in different pathologies, including cancer and is a current target for drug development. Bufadienolides are cytotoxic in tumor cells, but there is no enough evidences that inhibition of APN activity contributes to their effect. In the present contribution we investigated the effect of another member of the bufadienolide family, bufotalin, on porcine APN (pAPN) activity. Bufotalin inhibited pAPN activity with K
i
values in the submicromolar range and an uncompetitive inhibition mechanism; it also inhibited porcine aminopeptidase A (pAPA) activity, but with a classical reversible competitive inhibition mechanism. In addition, we determined the effect of bufotalin on the viability/metabolism of APN+ A549, H292, MeWo and CT26 cancer cells. Bufotalin was cytotoxic in a dose dependent manner; the highest cytotoxicity was detected in A549 cells, the cells with the highest APN activity. Thus, tumor cell line sensitivity to the cytotoxic effect of bufotalin correlates with cell surface APN activity.
Ectopeptidases are particularly interesting due to their potential to regulate/dysregulate the peptide mediated signaling cellular pathways because the active site located to the extracellular space. Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) is currently one of the ectopeptidases that has a great and complex influence on important physiological and pathological processes. Due to its influence on the immune system, type 2 diabetes mellitus, pulmonary pathologies, cardiovascular system, viral infections and cancer, DPP-IV is very attractive as a possible therapeutic target. However, its versatility makes such expectations very difficult. The aim of this work is to summarize relevant structural and functional aspects of DPP-IV and the role of this protein in several pathologies with special emphasis on cancer. DPP-IV role in cancer seems to depend on specific location, histologic type of tumour, tumour microenvironment, and presence/absence of molecules able to interact with DPP-IV. Because of DPP-IV controversial effects, generalizations are difficult and most of the time the role of DPP-IV must be analyzed case by case. However, new evidences in cell lines, animal models and clinical studies suggest that DPP-IV inhibitors open a promissory window through new therapeutic strategies against some cancers.
Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.
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