Yamile González

Characterization and comparative 3D modeling of CmPI-II, a novel ‘non-classical’ Kazal-type inhibitor from the marine snail Cenchritis muricatus (Mollusca)

Abstract The complete amino acid sequence obtained by electrospray ionization tandem mass spectrometry of the proteinase inhibitor CmPI-II isolated from Cenchritis muricatus is described. CmPI-II is a 5480-Da protein with three disulfide bridges that inhibits human neutrophil elastase (HNE) (K i 2.6±0.2 nM), trypsin (K i 1.1±0.9 nM), and other serine proteinases such as subtilisin A (K i 30.8±1.2 nM) and pancreatic elastase (K i 145.0±4.4 nM); chymotrypsin, pancreatic and plasma kallikreins, thrombin and papain are not inhibited. CmPI-II shares homology with the Kazal-type domain and may define a new group of ‘non-classical’ Kazal inhibitors according to its CysI-CysV disulfide bridge position. The 3D model of CmPI-II exhibits similar secondary structure characteristics to Kazal-type inhibitors and concurs with circular dichroism experiments. A 3D model of the CmPI-II/HNE complex provides a structural framework for the interpretation of its experimentally determined K i value. The model shows both similar and different contacts at the primary binding sites in comparison with the structure of turkey ovomucoid third domain (OMTKY3)/HNE used as template. Additional contacts calculated at the protease-inhibitor interface could also contribute to the association energy of the complex. This inhibitor represents an exception in terms of specificity owing to its ability to strongly inhibit elastases and trypsin.

A novel metallocarboxypeptidase-like enzyme from the marine annelid Sabellastarte magnifica--a step into the invertebrate world of proteases.

After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhibitor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N‐terminal and internal sequence homologies with M14 metallocarboxypeptidase‐like enzymes, as determined by MS and automated Edman degradation. The enzyme contains one atom of Zn per molecule, is activated by Ca2+ and is drastically inhibited by the metal chelator 1,10‐phenanthroline, as well as by excess Zn2+ or Cu2+, but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallocarboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase‐A synthetic substrates, such as those with hydrophobic residues at the C‐terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O‐like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metallocarboxypeptidases family (assignable to the M14A or pancreatic‐like subfamily) with a wider specificity that has not been described previously.

Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood) Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K m 55.7 μM) in an optimum pH of 7.1, and this activity is effectively retained until 50°C. CeSP remained stable in the presence of kosmotropic anions (PO4  3−, SO4  2−, and CH3COO−) or chaotropic cations (K+ and Na+). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

Infestin 1R, an intestinal subtilisin inhibitor from Triatoma infestans able to impair mammalian cell invasion by Trypanosoma cruzi

Infestins are Kazal-type serine protease inhibitors described in the midgut of Triatoma infestans, Chagas disease vector. Of all infestins, only infestin 1R (INF1R) does not control host blood coagulation, due to its inhibitory specificity for chymotrypsin-like proteases. We further investigated the effect of INF1R on cell infection by Trypanosoma cruzi. The importance of INF1R reactive site to inhibit T. cruzi cell invasion was confirmed using 1RSFTI, a synthetic cyclic peptide containing the inhibitor reactive site region hybridized to the Sunflower Trypsin Inhibitor-1 (SFTI-1). Our results suggest that INF1R efficiently inhibited parasite cell invasion. For the first time, a serine protease inhibitor, derived from T. infestans, was shown to impair cell invasion by T. cruzi, representing possible new target in parasite cell invasion.

Glycosaminoglycans Modify Elastase Action In Vitro and Enhance Elastase-Induced Cell Death in Cultured Fibroblasts

Human neutrophil elastase (HNE) has been shown to be involved on death of different cell types, including epithelial lung cells, which is related to several pulmonary diseases. Since HNE activity may be influenced by extracellular matrix (ECM) molecules such as glycosaminoglycans (GAGs), and fibroblasts are the most common ECM-producing cells of lung connective tissue, the aim of this work was to verify if HNE can induce fibroblast death and to study the enzyme modulation by GAGs. HNE-like activity was mimicked by using human neutrophils conditioned medium (NCM). Heparan sulfate and chondroitin 6-sulfate reduce the enzyme activity and modify its secondary structure. NCM reduced cell viability, and this effect was higher in the presence of those GAGs. NCM also increased DNA fragmentation, suggesting the occurrence of apoptosis, but without influence of GAGs. These results can contribute to the understanding of HNE modulation in physio- and pathological processes where this enzyme is involved.