Mouse skin peptidomic analysis of the hemorrhage induced by a snake venom metalloprotease.

Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.

Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins.

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.

Cleavage of proteoglycans, plasma proteins and the platelet-derived growth factor receptor in the hemorrhagic process induced by snake venom metalloproteinases.

Envenoming by viperid snakes results in a complex pattern of tissue damage, including hemorrhage, which in severe cases may lead to permanent sequelae. Snake venom metalloproteinases (SVMPs) are main players in this pathogenesis, acting synergistically upon different mammalian proteomes. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, induces severe local hemorrhage at pmol doses in a murine model. Our hypothesis is that in a complex scenario of tissue damage, HF3 triggers proteolytic cascades by acting on a partially known substrate repertoire. Here, we focused on the hypothesis that different proteoglycans, plasma proteins, and the platelet derived growth factor receptor (PDGFR) could be involved in the HF3-induced hemorrhagic process. In surface plasmon resonance assays, various proteoglycans were demonstrated to interact with HF3, and their incubation with HF3 showed degradation or limited proteolysis. Likewise, Western blot analysis showed in vivo degradation of biglycan, decorin, glypican, lumican and syndecan in the HF3-induced hemorrhagic process. Moreover, antithrombin III, complement components C3 and C4, factor II and plasminogen were cleaved in vitro by HF3. Notably, HF3 cleaved PDGFR (alpha and beta) and PDGF in vitro, while both receptor forms were detected as cleaved in vivo in the hemorrhagic process induced by HF3. These findings outline the multifactorial character of SVMP-induced tissue damage, including the transient activation of tissue proteinases, and underscore for the first time that endothelial glycocalyx proteoglycans and PDGFR are targets of SVMPs in the disruption of microvasculature integrity and generation of hemorrhage.

Interaction of Bothrops jararaca venom metalloproteinases with protein inhibitors.

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.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3ε and calmodulin.

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.

The novel leptospiral surface adhesin Lsa20 binds laminin and human plasminogen and is probably expressed during infection.

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.

Effect of drought and re-watering on fructan metabolism in Vernonia herbacea (Vell.) Rusby.

Vernonia herbacea (Vell.) Rusby, a native species from the Brazilian Cerrado, accumulates about 80% of fructans in the rhizophores, the underground reserve organs. Besides their role as reserve, fructans have been recognized as protective compounds against drought. This physiological function attributed to fructans seems consistent with the wide occurrence of these carbohydrates in the cerrado, a biome that undergoes seasonal drought. The aim of this work was to analyze fructan composition and the activities of the enzymes involved in fructan synthesis, sucrose:sucrose 1-frutosyltransferase (1-SST) and fructan:fructan 1-frutosyltransferase (1-FFT), and depolymerization, fructan 1-exohydrolase (1-FEH) in plants submitted to water suppression. The plants were divided into 3 groups receiving 3 treatments: daily watering (control), water suppression for 23 days (WS) and re-watering after 15 days (RW). Samples were taken at the beginning of the experiment (Time 0) and after 3, 7, 11, 15, 17 and 23 days of water suppression. 1-SST and 1-FFT activities increased at the beginning of the water restriction period, coinciding with a decrease in 1-FEH activity, the onset of the reduction in soil water potential and in leaf water potential. Increases in 1-FEH and invertase activities led to a high yield of reducing sugars at the 23rd day after water suppression, and together with 1-FFT, 1-FEH also seemed to act in the redistribution of fructan molecules after re-watering. The increase in reducing sugars and in the fructo-oligo:fructo-polysaccharide ratio were associated to the maintenance of rhizophore turgor. Considering that WS plants showed changes in fructan metabolism that favored water retention and absorption after re-watering, the occurrence of osmotic adjustment mechanisms is suggested, reinforcing the hypothesis of fructans as protective agents against abiotic stresses, such as drought.

The novel leptospiral surface adhesin Lsa20 binds laminin and human plasminogen and is probably expressed during infection

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.

New insights into the structural elements involved in the skin haemorrhage induced by snake venom metalloproteinases.

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.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation.

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 mum. 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.

Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.