Leptospirosis is an emerging infectious disease caused by pathogenic species of Leptospira. In this work, we report the cloning, expression, purification, and characterization of two predicted leptospiral outer membrane proteins, LIC11469 and LIC11030. The LIC11469 protein is well conserved among leptospiral strains, while LIC11030 was identified only in Leptospira interrogans. We confirmed by surface proteolysis of intact leptospires with proteinase K that these proteins are most likely new surface leptospiral proteins. The recombinant proteins were evaluated for their capacity to attach to extracellular matrix (ECM) components and to plasminogen. The leptospiral protein encoded by LIC11469, named Lsa20 (leptospiral surface adhesin of 20 kDa), binds to laminin and to plasminogen. The binding with both components was not detected when Lsa20 was previously denatured or blocked with anti-Lsa20 antibodies. Moreover, Lsa20 binding to laminin was also confirmed by surface plasmon resonance (SPR). Laminin competes with plasminogen for binding to Lsa20, suggesting the same ligand-binding site. Lsa20-bound plasminogen could be converted to enzymatically active plasmin, capable of cleaving plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Lsa20 was recognized by antibodies in confirmed-leptospirosis serum samples, suggesting that this protein is expressed during infection. Taken together, our results indicate that Lsa20 is a novel leptospiral adhesin that in concert with the host-derived plasmin may help the bacteria to adhere and to spread through the hosts.

Haemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and extravasation. This study analysed structural elements important for the interaction of four Bothrops jararaca SVMPs of different domain organisation and glycosylation levels with plasma and extracellular matrix proteins: HF3 (P-III class) is highly glycosylated and ~80 times more haemorrhagic than bothropasin (P-III class), which has a minor carbohydrate moiety; BJ-PI (P-I class) is not haemorrhagic and the DC protein is composed of disintegrin-like/cysteine-rich domains of bothropasin. HF3, bothropasin and BJ-PI showed different degradation profiles of fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel; however, only bothropasin degraded collagen I. In solid-phase binding assays HF3 and bothropasin interacted with fibrinogen, fibronectin, laminin, collagens I and VI; the DC protein bound only to collagens I and VI; however, no binding of BJ-PI to these proteins was detected. N-deglycosylation caused loss of structural stability of bothropasin and BJ-PI but HF3 remained intact, although its haemorrhagic and fibrinogenolytic activities were partially impaired. Nevertheless, N-deglycosylated HF3 bound with higher affinity to collagens I and VI, although its proteolytic activity upon these collagens was not enhanced. This study demonstrates that features of carbohydrate moieties of haemorrhagic SVMPs may play a role in their interaction with substrates of the extracellular matrix, and the ability of SVMPs to degrade proteins in vitro does not correlate to their ability to cause haemorrhage, suggesting that novel, systemic approaches are necessary for understanding the mechanism of haemorrhage generation by SVMPs.

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Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca(2+) -calmodulin (CaM) and 14-3-3ε, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 μM, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3ε with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca(2+) concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

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Snake venom metalloproteinases (SVMPs) play important roles in the local and systemic hemorrhage observed upon envenomation. In a previous study on the structural elements important for the activities of HF3 (highly hemorrhagic, P-III-SVMP), bothropasin (hemorrhagic, P-III-SVMP) and BJ-PI (non-hemorrhagic, P-I-SVMP), from Bothrops jararaca, it was demonstrated that they differ in their proteolysis profile of plasma and extracellular matrix proteins. In this study, we evaluated the ability of proteins DM43 and α2-macroglobulin to interfere with the proteolytic activity of these SVMPs on fibrinogen and collagen VI and with their ability to induce hemorrhage. DM43 inhibited the proteolytic activity of bothropasin and BJ-PI but not that of HF3, and was not cleaved the three proteinases. On the other hand, α2-macroglobulin did not inhibit any of the proteinases and was rather cleaved by them. In agreement with these findings, binding analysis showed interaction of bothropasin and BJ-PI but not HF3 to DM43 while none of the proteinases bound to α2-macroglobulin. Moreover, DM43 promoted partial inhibition of the hemorrhagic activity of bothropasin but not that of HF3. Our results demonstrate that metalloproteinases of B. jararaca venom showing different domain composition, glycosylation level and hemorrhagic potency show variable susceptibilities to protein inhibitors.

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5 degrees C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly beta-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide‐metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium‐dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 μm. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187‐stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187‐stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7148420: EP24.15 (uniprotkb:http://www.ebi.uniprot.org/entry/P52888) and Calmodulin (uniprotkb:http://www.ebi.uniprot.org/entry/P62161) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by surface plasmon resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7148437: EP24.15 (uniprotkb:http://www.ebi.uniprot.org/entry/P52888) and Calmodulin (uniprotkb:http://www.ebi.uniprot.org/entry/P62158) colocalize (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403) by surface plasmon resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0107) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7148406: Calmodulin (uniprotkb:http://www.ebi.uniprot.org/entry/P62161) binds (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) to EP24.15 (uniprotkb:http://www.ebi.uniprot.org/entry/P52888) by pull down (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0096)