Abstract:The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such a… Show more
“…Sea anemone (phylum Cnidaria, class Anthozoa) venom is a rich source of proteinaceous toxins acting on ion channels such as voltage-gated sodium and potassium channels, and the TRPV1 [1,2,3,4,5] as well as a lot of protease inhibitors belonging, mainly, to a numerous BPTI/Kunitz-type family [6]. The peptides of this family have an ancient Kunitz fold and some of them are characterized by a unique and intriguing feature of dual functionality since they inhibit both proteases and ion channels [7,8,9,10].…”
Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10−7 and 7.0 × 10−7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.
“…Sea anemone (phylum Cnidaria, class Anthozoa) venom is a rich source of proteinaceous toxins acting on ion channels such as voltage-gated sodium and potassium channels, and the TRPV1 [1,2,3,4,5] as well as a lot of protease inhibitors belonging, mainly, to a numerous BPTI/Kunitz-type family [6]. The peptides of this family have an ancient Kunitz fold and some of them are characterized by a unique and intriguing feature of dual functionality since they inhibit both proteases and ion channels [7,8,9,10].…”
Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10−7 and 7.0 × 10−7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.
“…Consequently, a distinct disulfide bridge may be generated [22,39]. Additionally, among other scorpion protease inhibitors, recombinant BmKTT-2 was found to possess eight cysteine residues connected by four disulfide bridges, which is different architectural property from all known Kunitz-type animal toxins [11,39] (Figure 1). …”
“…Nevertheless, rLmKTT-1b along with the scorpion peptides rLmKTT-1a, rLmKTT-1c, and rBmKTT-1 [11,39] contain a unique cysteine framework, different from the classical Kunitz-type motif, where the normal C2-C4 disulfide bridge is absent, but two additional cysteine residues are present at the C-terminus of the mature peptide, which might create a new disulfide bond. Consequently, a distinct disulfide bridge may be generated [22,39].…”
“…Nevertheless, rLmKTT-1b along with the scorpion peptides rLmKTT-1a, rLmKTT-1c, and rBmKTT-1 [11,39] contain a unique cysteine framework, different from the classical Kunitz-type motif, where the normal C2-C4 disulfide bridge is absent, but two additional cysteine residues are present at the C-terminus of the mature peptide, which might create a new disulfide bond. Consequently, a distinct disulfide bridge may be generated [22,39]. Additionally, among other scorpion protease inhibitors, recombinant BmKTT-2 was found to possess eight cysteine residues connected by four disulfide bridges, which is different architectural property from all known Kunitz-type animal toxins [11,39] (Figure 1).…”
“…They have different structural scaffolds regarding their pharmacological targets. Active principles binding on ionic channel are characterized by three beta strands [7], while those binding on enzymes such as the family of Kunitz-type inhibitors have two beta strands and an alpha helix [8]. Surprisingly the sea anemone toxin Bg1 can compete at nano molar concentrations on the same pharmacological site on sodium channel than the Aah II scorpion toxin with a scaffold completely different [9].…”
Sea anemones are a remarkable source of active principles due to a decentralized venom system. Blood vessel formation or angiogenesis is a very promising target against cancer, but the few available antiangiogenic compounds have a limited efficacy. In this study, a protein fraction was purified from tentacles of Anemonia viridis able to limit endothelial cells proliferation and vessel network formation or angiogenesis at low concentration (14 nM). The sequences in this protein fraction were determined with Edman degradation and Mass Spectrometry In Source Decay and revealed homologies with BDS sea anemones. The presence of a two turn alpha helix observed with Circular Dichroism and a trypsin activity inhibition suggested that the active principle could be a Kunitz-type inhibitor, which may interact with an integrin due to a RGD motif well exposed to the solvent as revealed by Molecular Modeling. This active principle could improve antiangiogenic therapy from existing antiangiogenic compounds binding on the VEGF.
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