Beatriz L. Fernandes

The C-type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin-converting enzyme

The bradykinin‐potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin‐converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin‐converting enzyme in blood pressure regulation. In the present study, seven bradykinin‐potentiating peptides were identified within the C‐type natriuretic peptide precursor cloned from snake brain. The bradykinin‐potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin‐converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin‐potentiating peptides, one of which displays high specificity toward the N‐domain active site of the somatic angiotensin‐converting enzyme. In situ hybridization studies revealed the presence of the bradykinin‐potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin‐potentiating peptides, their presence within the neuroendocrine regulator C‐type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.

Administration of M. leprae Hsp65 Interferes with the Murine Lupus Progression

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K409A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F1 mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animals life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K409A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K409A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.

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 (<EKWAP), BPP-7a (<EDGPIPP), BPP-9a (<EWPRPQIPP) and BPP-12b (<EWGRPPGPPIPP) - and studied their stability when exposed to the action of endogenous animal proteolytic enzymes. Here we characterized the BPP metabolites in mouse urine by MALDI-TOF mass spectrometry. Biotransformation results indicated the following: BPP-7a showed a higher resistance to proteolytic cleavage; BPP-5a is metabolized in tripeptides (<EKW); BPP-9a was identified in the intact form and in the form of two metabolites (<EWPRP; <EWPRPQIP); and BPP-12a proved to be very susceptible to hydrolysis by proteolytic enzymes. Thus, the results obtained support the hypothesis that diverse biological functions for each BPP could be mediated by different interactions with alternative targets, and not only by sACE inhibition.

A Mycobacterium leprae Hsp65 Mutant as a Candidate for Mitigating Lupus Aggravation in Mice

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F1 mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K409A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K409A pep synthetic peptides, which cover residues 352–371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K409A+Leader pep or K409A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352–371 region. The number of interactions observed for WT is much higher than for Hsp65 K409A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F1 mice were inoculated with Hsp60 or K409A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K409A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.