Insights into cardiovascular effects of proline-rich oligopeptide (Bj-PRO-10c) revealed by structure-activity analyses: dissociation of antihypertensive and bradycardic effects.

We have previously reported that the proline-rich decapeptide from Bothrops jararaca (Bj-PRO-10c) causes potent and sustained antihypertensive and bradycardic effects in SHR. These activities are independent of ACE inhibition. In the present study, we used the Ala-scan approach to evaluate the importance of each amino acid within the sequence of Bj-PRO-10c (Pyr(1)-Asn(2)-Trp(3)-Pro(4)-His(5)-Pro(6)-Gln(7)-Ile(8)-Pro(9)-Pro(10)). The antihypertensive and bradycardic effects of the analogues Bj-PRO-10c Ala(3), Bj-PRO-10c Ala(7), Bj-PRO-10c Ala(8) were similar to those of Bj-PRO-10c, whereas the analogues Bj-PRO-10c Ala(2), Bj-PRO-10c Ala(4), Bj-PRO-10c Ala(5), Bj-PRO-10c Ala(9), and Bj-PRO-10c Ala(10) kept the antihypertensive activity and lost bradycardic activity considerably. In contrast, Bj-PRO-10c Ala(1) and Bj-PRO-10c Ala(6) were unable to provoke any cardiovascular activity. In summary, we demonstrated that (1) the Pyr(1) and Pro(6) residues are essential for both, the antihypertensive and bradycardic effects of Bj-PRO-10c; (2) Ala-scan approach allowed dissociating blood pressure reduction and bradycardic effects. Conformational properties of the peptides were examined by means of circular dichroism (CD) spectroscopy. The different Ala-scan analogues caused either an increase or decrease in the type II polyproline helix content compared to Bj-PRO-10c. The complete loss of activity of the Pro(6) â†’ Ala(6) mutant is probably due to the fact that in the parent peptide the His(5)-Pro(6) bond can exist in the cis configuration, which could correspond to the conformation of this bond in the bound state. Current data support the Bj-PRO-10c as a promising leader prototype to develop new agents to treat cardiovascular diseases and its co-morbidities.

Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes.

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.

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 snake venom peptide Bj-PRO-7a is a M1 muscarinic acetylcholine receptor agonist.

Proline-rich peptides from Bothrops jararaca venom (Bj-PRO) were characterized based on the capability to inhibit the somatic angiotensin-converting enzyme. The pharmacological action of these peptides resulted in the development of Captopril, one of the best examples of a target-driven drug discovery for treatment of hypertension. However, biochemical and biological properties of Bj-PROs were not completely elucidated yet, and many recent studies have suggested that their activity relies on angiotensin-converting enzyme-independent mechanisms. Here, we show that Bj-PRO-7a (

Assessing the role of endooligopeptidase activity of Ndel1 (nuclear-distribution gene E homolog like-1) in neurite outgrowth.

Ndel1 plays multiple roles in neuronal development but it is unknown whether its reported cysteine protease activity is important for these processes. Ndel1 is known to be critical for neurite outgrowth in PC12 cells where it works co-operatively in a complex with DISC1 to allow normal neuritogenesis. Through an initial interest in understanding the regulation of the expression of Ndel1 during neuronal differentiation, we have been able to show that Ndel1 expression and enzyme activity is up-regulated during neurite outgrowth in PC12 cells induced to neural differentiation. Heterologous expression of wild-type Ndel1 (Ndel1(WT)) in PC12 cells increases the percentage of cells bearing neurites in contrast to the catalytically dead mutant, Ndel1(C273A), which caused a decrease. Furthermore depletion of endogenous Ndel1 by RNAi decreased neurite outgrowth, which was rescued by transfection of the enzymatically active Ndel1(WT), but not by the Ndel1(C273A) mutant. Together these data support the notion that the endooligopeptidase activity of Ndel1 plays a crucial role in the differentiation process of PC12 cells to neurons. Genetic data and protein interaction with DISC1 might suggest a role for Ndel1 in neuropsychiatirc conditions.

Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey.

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

The central nervous system as target for antihypertensive actions of a proline-rich peptide from Bothrops jararaca venom.

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (

Analysis of the subproteomes of proteinases and heparin-binding toxins of eight Bothrops venoms.

Viperid snakes show the most complex snake-venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2-DE, and their subproteomes of proteinases were explored by 2-D immunostaining and 2-D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti-coagulant and anti-inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high-affinity for heparin as composed of mainly serine proteinases and C-type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin-binding toxins. Only the non-bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin-bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.

Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility.

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg(-1) reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 microM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 microM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 microM inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine.

Activation of leukocyte rolling by the cysteine-rich domain and the hyper-variable region of HF3, a snake venom hemorrhagic metalloproteinase.

The functionality of the disintegrin-like/cysteine-rich domains of snake venom metalloproteinases (SVMPs) has been shown to reside in the cysteine-rich region, which can interact with VWA-containing proteins. Recently, the hyper-variable region (HVR) of the cysteine-rich domain was suggested to constitute a potential protein-protein adhesive interface. Here we show that recombinant proteins of HF3, a hemorrhagic P-III SVMP, containing the cysteine-rich domain (disintegrin-like/cysteine-rich and cysteine-rich proteins) but not the disintegrin-like protein were able to significantly increase leukocyte rolling in the microcirculation. Peptides from the HVR also promoted leukocyte rolling and this activity was inhibited by anti-alpha(M)/beta2 antibodies. These results show, for the first time, that the cysteine-rich domain and its HVR play a role in triggering pro-inflammatory effects mediated by integrins.

A novel bradykinin potentiating peptide isolated from Bothrops jararacussu venom using catallytically inactive oligopeptidase EP24.15.

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. Here, we use a substrate-capture assay that employs a catalytically inactive mutant of thimet oligopeptidase (EC 3.4.24.15; EP24.15) to identify novel bioactive peptides in Bothrops jararacussu venom. Of the peptides captured with inactive EP24.15 and identified by mass spectrometry, three were previously identified bradykinin-potentiating peptides (BPP),

Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus.

We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of kappa- and delta-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be

A novel physiological property of snake bradykinin-potentiating peptides-reversion of MK-801 inhibition of nicotinic acetylcholine receptors.

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [

Characterization of urinary metabolites from four synthetic bradykinin potentiating peptides (BPPs) in mice.

BPPs have been identified in the venom of the Bothrops jararaca snake, or deduced from precursor proteins expressed either in the venom gland or in the brain of the snake. Their potentiating activity on bradykinin (Bk) is assumed to occur through a somatic angiotensin-converting enzyme (sACE) inhibitory mechanism. We have demonstrated that synthetic BPPs show remarkable functional differences, despite their high amino acid sequence similarities. Recently, we demonstrated that BPP-10c, after i.p. administration, was found in its intact form and in the form of a unique metabolite (des-Pro(10) BPP-10c) in mouse urine. Given this finding, we selected a number of BPPs with different structure-activities - BPP-5a (

Tissue distribution in mice of BPP 10c, a potent proline-rich anti-hypertensive peptide of Bothrops jararaca.

The snake venom proline-rich peptide BPP 10c is an active somatic angiotensin-converting enzyme (sACE) inhibitors. Recently we demonstrated that the anti-hypertensive effect of BPP 10c is not related to the inhibition of sACE alone, thus suggesting that this enzyme is not its only target for blood pressure reduction. In the present work, a biodistribution study in Swiss mice of [(125)I]-BPP 10c in the absence or in the presence of a saturating concentration of captopril, a selective active-site inhibitor of sACE, demonstrated that: (1) [(125)I]-BPP 10c was present in several organs and the renal absorption was significantly high; (2) [(125)I]-BPP 10c showed a clear preference for the kidney, maintaining a high concentration in this organ in the presence of captopril for at least 3h; (3) The residual amount of [(125)I]-BPP 10c in the kidney of animals simultaneously treated with captopril suggest that the peptide can interact with other targets different from sACE in this organ. We also showed that Cy3-labeled BPP 10c was internalized by human embryonic kidney cells (HEK-293T). Taken together, these results suggest that sACE inhibition by captopril affects the tissue distribution of [(125)I]-BPP 10c and that the anti-hypertensive effects of BPP 10c are not only dependent on sACE inhibition.

Identification of novel bradykinin-potentiating peptides (BPPs) in the venom gland of a rattlesnake allowed the evaluation of the structure-function relationship of BPPs.

Aiming to extend the knowledge about the diversity of bradykinin-potentiating peptides (BPPs) and their precursor proteins, a venom gland cDNA library from the South American rattlesnake (Crotalus dursissus terrificus, Cdt) was screened. Two novel homologous cDNAs encoding the BPPs precursor protein were cloned. Their sequence contain only one single longer BPP sequence with the typical IPP-tripeptide, and two short potential BPP-like molecules, revealing a unique structural organization. Several peptide sequences structurally similar to the BPPs identified in the precursor protein from Cdt and also from others snakes, were chemically synthesized and were bioassayed both in vitro and in vivo, by means of isolated smooth muscle preparations and by measurements of blood pressure in anaesthetized rats, respectively. We demonstrate here that a pyroglutamyl residue at the N-terminus with a high content of proline residues, even with the presence of a IPP moiety characteristic of typical BPPs, are not enough to determine a bradykinin-potentiating activity to these peptides. Taken together, our results indicate that the characterization of the BPPs precursor proteins and identification of characteristic glutamine residues followed by proline-rich peptide sequences are not enough to predict if these peptides, even with a pyroglutamyl residue at the N-terminus, will present the typical pharmacological activities described for the BPPs.

Novel pharmaceutical composition of bradykinin potentiating penta peptide with beta-cyclodextrin: physical-chemical characterization and anti-hypertensive evaluation.

This work describes chemical properties and anti-hypertensive activity of an oral pharmaceutical formulation obtained from the complexation of beta-cyclodextrin (beta-CD) with bradykinin potentiating penta peptide (BPP-5a) founded in the Bothrops jararaca poison. Physical chemistry characterizations were recorded in order to investigate the intermolecular interactions between species in complex. Circular dichroism data indicated conformational changes of BPP-5a upon complexation with beta-CD. ROESY and theoretical calculations showed a selective approximation of triptophan moiety into cavity of beta-CD. Isothermal titration calorimetry data indicated an exothermic formation of the complex, which is accomplished by reduction of entropy. The anti-hypertensive activity of the BPP-5a/beta-CD complex has been evaluated in spontaneous hypertensive rats, showing better results than pure BPP-5a.

Cloning and characterization of the human and rabbit NUDEL-oligopeptidase promoters and their negative regulation.

NUDEL-oligopeptidase is a cytosolic cysteine peptidase, active towards oligopeptides and involved in the conversion and inactivation of a number of bioactive peptides. This protein interacts with neuronal proteins and is essential for brain development and cortical organization during embryogenesis. In this study, 5'-flanking sequences of the human and rabbit NUDEL-oligopeptidase gene were cloned into the pGL3 reporter gene vector and the promoter activity of the full-length fragment and deletions series was measured in transient transfection assays using two different cell lines, namely, C6 rat glioma and NH15 human neuroblastoma. Overall, a very similar pattern of promoter activity was obtained for both rabbit and human NUDEL-oligopeptidase promoter sequences, and their respective serial deletion constructs upon transient transfection into these cell lines. The only exception was for the longest rabbit upstream sequence that displayed about 1.8-fold higher luciferase expression upon transfection into NH15 neuronal cells than that observed upon transfection into C6 glioma cells. On the other hand, no significant difference was observed for the human longest sequence. These results are in good agreement with the expression pattern of NUDEL-oligopeptidase in human and rabbit tissues.

Sex-based individual variation of snake venom proteome among eighteen Bothrops jararaca siblings.

Variation of venom proteome is relevant to basic research, to management of envenoming, and to studies on the evolution of poisonous snakes. In this study, we explored the venom proteomes of eighteen Bothrops jararaca specimens of a single litter born and raised in laboratory. Using electrophoretic techniques and various protocols for measuring the proteolytic activities of these venoms we have detected individual variability and highlighted sex-specific proteomic similarities and differences among sibling snakes. SDS-polyacrylamide gel electrophoresis under non-reducing conditions showed protein bands of approximately 100 kDa specific of male venoms. 2D-electrophoresis showed regions with varying spot complexity between pooled female and male venoms as well as spots that were gender specific. Gelatin zymography showed that female venoms contained proteinases of approximately 25 kDa absent from male venoms. Female venoms were more active than male venoms in degrading fibrinogen whereas on fibrin no significant differences were detected. Among various chromogenic peptide substrates tested, male venoms showed higher amidolytic activity than female venoms on D-Val-Leu-Lys-pNA and D-Phe-Pip-Arg-pNA. Taken together, these results show sex-based differences in the venom proteome of sibling snakes of a single litter raised under controlled conditions which seem to be genetically inherited and imposed by evolutionary forces.

The unusual high molecular mass of Bothrops protease A, a trypsin-like serine peptidase from the venom of Bothrops jararaca, is due to its high carbohydrate content.

Bothrops protease A (BPA) is a serine peptidase isolated from the venom of Bothrops jararaca. Unlike many venom enzymes, it is stable at pHs between 3 and 9 and resists heating at 86 degrees C for 10 min. Mature snake venom serine peptidases of the chymotrypsin family are in general glycoproteins composed of around 232 amino acids and their molecular masses vary between 25 and 40 kDa. BPA is a glycosylated protein that migrates on SDS-polyacrylamide gel electrophoresis (PAGE) as a single band of 67 kDa. In order to find out whether BPA has the typical serine peptidase primary structure or if it is composed of a longer amino acid sequence, we cloned a cDNA encoding BPA. Its deduced amino acid sequence showed that BPA is composed of 234 residues with a calculated molecular mass of 25,409 Da implying that approximately 62% of its molecular mass assessed by SDS-PAGE is due to carbohydrate moieties. Eight putative N-glycosylation and two putative O-glycosylation sites were found in BPA amino acid sequence. Deglycosylation experiments indicated that all 10 potential glycosylation sites in BPA are utilized. Complete N- and O-deglycosylation was only achieved under denaturing conditions and generated main products of 25 and 55 kDa, respectively, which were enzymatically inactive. N-deglycosylation under non-denaturing conditions was only partial and gave a main product of 50 kDa and fragments ranging from 25 to approximately 10 kDa. Kinetic parameters K(m) and V(max) of partially N-deglycosylated BPA upon substrate Bz-Arg-pNA were similar to the native form. However, when partially N-deglycosylated BPA was submitted to pH 3 and pH 10, it appeared to be unstable as it underwent hydrolysis, as shown by the presence of two main products of 30 and 12 kDa while the 50 kDa protein band disappeared. These changes also had effects on V(max) upon Bz-Arg-pNA which dropped to approximately 45%, while K(m) values remained unchanged. Fluorescence emission spectroscopy indicated that in partially N-deglycosylated BPA, tryptophan residues are more exposed to a polar environment than in the fully glycosylated protein. Taken together, these studies indicate that glycosylation has a stabilizing effect on BPA.