Kininogenase Activity Research Articles

BmooMPα-I, a Metalloproteinase Isolated from Bothrops moojeni Venom, Reduces Blood Pressure, Reverses Left Ventricular Remodeling and Improves Cardiac Electrical Conduction in Rats with Renovascular Hypertension.

BmooMPα-I has kininogenase activity, cleaving kininogen releasing bradykinin and can hydrolyze angiotensin I at post-proline and aspartic acid positions, generating an inactive peptide. We evaluated the antihypertensive activity of BmooMPα-I in a model of two-kidney, one-clip (2K1C). Wistar rats were divided into groups: Sham, who underwent sham surgery, and 2K1C, who suffered stenosis of the right renal artery. In the second week of hypertension, we started treatment (Vehicle, BmooMPα-I and Losartan) for two weeks. We performed an electrocardiogram and blood and heart collection in the fourth week of hypertension. The 2K1C BmooMPα-I showed a reduction in blood pressure (systolic pressure: 131 ± 2 mmHg; diastolic pressure: 84 ± 2 mmHg versus 174 ± 3 mmHg; 97 ± 4 mmHg, 2K1C Vehicle, p < 0.05), improvement in electrocardiographic parameters (Heart Rate: 297 ± 4 bpm; QRS: 42 ± 0.1 ms; QT: 92 ± 1 ms versus 332 ± 6 bpm; 48 ± 0.2 ms; 122 ± 1 ms, 2K1C Vehicle, p < 0.05), without changing the hematological profile (platelets: 758 ± 67; leukocytes: 3980 ± 326 versus 758 ± 75; 4400 ± 800, 2K1C Vehicle, p > 0.05), with reversal of hypertrophy (left ventricular area: 12.1 ± 0.3; left ventricle wall thickness: 2.5 ± 0.2; septum wall thickness: 2.3 ± 0.06 versus 10.5 ± 0.3; 2.7 ± 0.2; 2.5 ± 0.04, 2K1C Vehicle, p < 0.05) and fibrosis (3.9 ± 0.2 versus 7.4 ± 0.7, 2K1C Vehicle, p < 0.05). We concluded that BmooMPα-I improved blood pressure levels and cardiac remodeling, having a cardioprotective effect.

Nomenclature of factor XI and the contact system

Nomenclature of factor XI and the contact system

C1 inhibitor function using contact-phase proteases as target: evaluation of an innovative assay.

Controlling prekallikrein activation by C1 inhibitor (C1Inh) represents the most essential mechanism for angioedema patient protection. C1Inh function in the plasma is usually measured based on the residual activity of the C1s protease not involved in the pathological process. We have hereby proposed an alternative enzymatic measurement of C1Inh function based on contact-phase activation and correlation with angioedema diagnostic requirements. The contact phase was reconstituted using the purified components, with C1Inh standard or plasma sample. The kinetics of the amidase activity were monitored using Pro-Phe-Arg-pNA, independently of alpha2-macroglobulin. We prevented any interference from a possible high plasma kininogenase activity by preincubating the samples with protease inhibitor. Receiver operating characteristics (ROC) were used to calculate the assay's diagnostic performance. The calibration curve was built using C1Inh standard (threshold limit 0.10×10(-3) U, i.e., 0.2pmol), and C1Inh function was quantified in the sample, with a reference interval established based on healthy individuals (n=281; men: 0.61-1.10U/ml, median: 0.85U/ml; women: 0.42-1.08U/ml, median: 0.74U/ml). The median values of female donors were lower than those of the others due to estrogen, yet C1Inh function remained within the reference interval. The ROC curve calculation provided the following optimum diagnostic cutoff values: women 0.36U/ml (area under curve [AUC]: 0.99; sensitivity: 93.48%; specificity: 99.37%); and men 0.61U/ml (AUC: 1; sensitivity: 100.0%; specificity: 100.0%). The performance outcome provided features suitable for angioedema diagnostic or follow-up. Established by means of the kinin formation process, this assay should be preferred over the method based on a C1s protease target.

Bioactive Components in Fish Venoms

Animal venoms are widely recognized excellent resources for the discovery of novel drug leads and physiological tools. Most are comprised of a large number of components, of which the enzymes, small peptides, and proteins are studied for their important bioactivities. However, in spite of there being over 2000 venomous fish species, piscine venoms have been relatively underrepresented in the literature thus far. Most studies have explored whole or partially fractioned venom, revealing broad pharmacology, which includes cardiovascular, neuromuscular, cytotoxic, inflammatory, and nociceptive activities. Several large proteinaceous toxins, such as stonustoxin, verrucotoxin, and Sp-CTx, have been isolated from scorpaenoid fish. These form pores in cell membranes, resulting in cell death and creating a cascade of reactions that result in many, but not all, of the physiological symptoms observed from envenomation. Additionally, Natterins, a novel family of toxins possessing kininogenase activity have been found in toadfish venom. A variety of smaller protein toxins, as well as a small number of peptides, enzymes, and non-proteinaceous molecules have also been isolated from a range of fish venoms, but most remain poorly characterized. Many other bioactive fish venom components remain to be discovered and investigated. These represent an untapped treasure of potentially useful molecules.

Exploration biologique des angiœdèmes à kinines

Kinin-mediated angioedema results from accumulation of kinins, vasoactive and vasopermeant peptides, on the vascular endothelium. The disease is characterized by sudden episodes of swelling in the subcutaneous and submucosal tissues; the edema may occur spontaneously or it may be precipitated by triggering factors such as physical or emotional stress, or certain medicines. The characterization of kinin formation and catabolism systems helps improve knowledge of the aetiopathogenic mechanisms involved and provides the basis for classification of kinin-mediated angioedema conditions; thus, we may distinguish between angioedema with C1 inhibitor deficiency, whether inherited or acquired, and angioedema with normal C1 inhibitor activity, associated with increased kinin-forming activity or deficiency in kinin catabolism enzymes. In support of the clinical diagnosis, the physician may request laboratory investigation for a functional and molecular definition of the disease. Laboratory diagnosis is based on the characterization of: (1) kinin production control by C1 inhibitor investigation (function, antigen levels and circulating species); (2) kinin production (kininogenase activity, kininogen cleavage species); and (3) kinin catabolism enzymes (aminopeptidase P, carboxypeptidase N, angiotensin-I converting enzyme and dipeptidyl peptidase IV). An abnormal biological phenotype is supported by examination of susceptibility genes (SERPING1, F12 and XPNPEP2) and mutation segregation in the families.

Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity

KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F, Y or M, and its S1′ and S2′ subsites showed selectivity for hydrophilic amino acids, particularly R and K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz–MISLM↓KRPPGFSPF↓RSSRI-NH2 (↓indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. The peptide Abz-NLY↓RVE-Q-EDDnp is the best synthetic substrate so far described for KLK7 [kcat/Km=455 (mMs)−1] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (Ki=33mM) and is present in skin moisturizing factor (124mM). The KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: Insights into substrate selectivity and kinetic behavior

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2–S′2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

Enzymatic Assays for the Diagnosis of Bradykinin-Dependent Angioedema

BackgroundThe kinins (primarily bradykinin, BK) represent the mediators responsible for local increase of vascular permeability in hereditary angioedema (HAE), HAE I-II associated with alterations of the SERPING1 gene and HAE with normal C1-Inhibitor function (HAE-nC1INH). Besides C1-Inhibitor function and concentration, no biological assay of kinin metabolism is actually available to help physicians for the diagnosis of angioedema (AE). We describe enzymatic tests on the plasma for diagnosis of BK-dependent AE.MethodsThe plasma amidase assays are performed using the Pro-Phe-Arg-p-nitroanilide peptide substrate to evaluate the spontaneous amidase activity and the proenzyme activation. We analyzed data of 872 patients presenting with BK-dependent AE or BK-unrelated diseases, compared to 303 controls. Anti-high MW kininogen (HK) immunoblot was achieved to confirm HK cleavage in exemplary samples. Reproducibility, repeatability, limit of blank, limit of detection, precision, linearity and receiver operating characteristics (ROC) were used to calculate the diagnostic performance of the assays.ResultsSpontaneous amidase activity was significantly increased in all BK-dependent AE, associated with the acute phase of disease in HAE-nC1INH, but preserved in BK-unrelated disorders. The increase of the amidase activity was associated to HK proteolysis, indicating its relevance to identify kininogenase activity. The oestrogens, known for precipitating AE episodes, were found as triggers of enzymatic activity. Calculations from ROC curves gave the optimum diagnostic cut-off for women (9.3 nmol⋅min−1⋅mL−1, area under curve [AUC] 92.1%, sensitivity 80.0%, and specificity 90.1%) and for men (6.6 nmol·min−1⋅mL−1, AUC 91.0%, sensitivity 87.0% and specificity 81.2%).ConclusionThe amidase assay represents a diagnostic tool to help physicians in the decision to distinguish between BK-related and –unrelated AE.

Pro-angiogenic effect of human kallikrein-related peptidase 12 (KLK12) in lung endothelial cells does not depend on kinin-mediated activation of B2 receptor

Kallikrein-12 (KLK12) may play an important role in angiogenesis modulating proangiogenic factor bioavailability and activating the kinin receptor B2 pathway. We studied whether KLK12 had an impact on angiogenesis and the activation of kinin receptor B2 results from the KLK12-dependent generation of kinins. KLK12 efficiently hydrolyzed high molecular weight kininogen, liberating a fragment containing the carboxy-terminal end of kinins. The kininogenase activity of KLK12 was poor, however, due to the cleavage resistance of the N-terminal side of the kinin sequence. A very low amount of kinins was accordingly released after in vitro incubation of high molecular weight kininogen with KLK12 and thus the proangiogenic activity of KLK12 in lung endothelial cells was not related to a kinin release.

Substrate specificity studies of the cysteine peptidases falcipain-2 and falcipain-3 from Plasmodium falciparum and demonstration of their kininogenase activity

We studied the substrate specificity requirements of recombinant cysteine peptidases from Plasmodium falciparum, falcipain-2 (FP-2) and falcipain-3 (FP-3), using fluorescence resonance energy transfer (FRET) peptides as substrates. Systematic modifications were introduced in the lead sequence Abz-KLRSSKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp=N-[2,4-dinitrophenyl]ethylenediamine) resulting in five series assayed to map S3−S′2 subsite specificity. Despite high sequence identity and structural similarity between FP-2 and FP-3, noteworthy differences in substrate specificity were observed. The S1 subsite of FP-2 preferentially accommodates peptides containing the positively charged residue Arg in P1, while FP-3 has a clear preference for the hydrophobic residue Leu in this position. The S2 subsite of FP-2 and FP-3 presents a strict specificity for hydrophobic residues, with Leu being the residue preferred by both enzymes. FP-2 did not show preference for the residues present at P3, while FP-3 hydrolysed the peptide Abz-ALRSSRQ-EDDnp, containing Ala at P3, with the highest catalytic efficiency of all series studied. FP-2 has high susceptibility for substrates containing hydrophobic residues in P′1, while FP-3 accommodates well peptides containing Arg in this position. The S′2 subsite of both enzymes demonstrated broad specificity. In addition, radioimmunoassay experiments indicated that kinins can be generated by FP-2 and FP-3 proteolysis of high molecular weight kininogen (HK). Both enzymes excised Met-Lys-bradykinin, Lys-bradykinin and bradykinin from the fluorogenic peptide Abz-MISLMKRPPGFSPFRSSRI-NH2, which corresponds to the Met375 to Ile393 sequence of HK. The capability of FP-2 and FP-3 to release kinins suggests the involvement of these enzymes in the modulation of malaria pathophysiology.

Angio-Oedema Induced by Oestrogen Contraceptives Is Mediated by Bradykinin and Is Frequently Associated with Urticaria

Background: Hereditary C1-inhibitor (C1-Inh) deficiency is associated with ‘bradykinin-mediated angio-oedema’ (BK-AO) and is believed not to be associated with urticaria. Acquired AO has been related to oestrogen contraceptives. Objective: To demonstrate that AO precipitated by oestrogens and characterized by nonfunctional C1-Inh is mediated by BK and to evaluate the occurrence of urticaria in these patients. Methods: A retrospective evaluation of patients referred for AO related to oestrogen was undertaken. Circulating C1-Inh, high molecular weight kininogen (HK) and enzymes involved in the metabolism of bradykinin were investigated. Results: Fifteen patients were included. HK cleavage concurrent to oestrogen intake was demonstrated in 10 patients with available plasma. Eight patients reported recurrent or chronic urticaria. Discontinuation of the contraceptive resulted in a return to native C1-Inh and HK in all cases studied and to normal kininogenase activity in all but one. The clinical manifestations completely disappeared in 6 patients and improved in 7 after the withdrawal of oestrogen. Conclusion: Patients display extensive cleavage of HK in the plasma, which supports that AO precipitated by oestrogen contraception is BK-mediated. Recurrent urticaria may have been underestimated in this context. The presence of recurrent urticaria should not systematically rule out the diagnosis of BK-AO when the history is suggestive.

Extracellular aspartic protease SAP2 of Candida albicans yeast cleaves human kininogens and releases proinflammatory peptides, Met-Lys-bradykinin and des-Arg9-Met-Lys-bradykinin

Bradykinin-related peptides, universal mediators of inflammation collectively referred to as the kinins, are often produced in excessive amounts during microbial infections. We have recently shown that the yeast Candida albicans, the major fungal pathogen to humans, can exploit two mechanisms to enhance kinin levels at the sites of candidial infection, one depending on adsorption and activation of the endogenous kinin-generating system of the host on the fungal cell wall and the other relying on cleavage of kinin precursors, the kininogens, by pathogen-secreted proteases. This work aimed at assigning this kininogenase activity to the major secreted aspartic protease of C. albicans (SAP2). The purified SAP2 was shown to cleave human kininogens, preferably the low molecular mass form (LK) and optimally in an acidic environment (pH 3.5-4.0), and to produce two kinins, Met-Lys-bradykinin and its derivative, [Hydroxyproline(3)]-Met-Lys-bradykinin, both of which are capable of interacting with cellular bradykinin receptors of the B2 subtype. Additionally, albeit with a lower yield, des-Arg(9)-Met-Lys-bradykinin, an effective agonist of B1-subtype receptors, was released. The pathophysiological potential of these kinins and des-Arg-kinin was also proven by presenting their ability to stimulate human promonocytic cells U937 to release proinflammatory interleukin 1β (IL-1β) and IL-6.

Local inflammatory response induced by scorpionfish Scorpaena plumieri venom in mice

The Scorpaena plumieri fish venom induces a severe pain and edema, observed both clinically and experimentally. In order to understand more about the envenomation syndrome, the present study characterized experimentally the local acute inflammatory response induced by S. plumieri venom (SpV) in a mouse model of tissue injury. Our results demonstrated that the local inflammatory response provoked after 2 h of SpV injection in footpad of mice is characterized by release of pivotal pro-inflammatory mediators (TNF, IL-6 and MCP-1). These mediators could be associated with histopathological changes observed into paw tissue, characterized by cellular infiltration, mainly neutrophils. Additionally, an investigation of edema formation pathways involved in inflammatory response was performed. SpV-induced edema was reduced significantly by previous administration of aprotinin or icatibant (HOE-140). However, the pre-treatment with diclofenac sodium and promethazine had less effect on this response. These results demonstrate that the kallikrein-kinin system (KKS) plays a major role in the edema formation. Despite the whole venom hydrolyzed the kallikrein synthetic substrate S-2302 (Pro-Phe-Arg-pNA), its main pro-inflammatory fraction was devoid of kininogenase activity. Our results demonstrate that SpV evokes a complex inflammatory reaction stimulating a secretion of TNF, IL-6, MCP-1 and leukocytes recruitment at the site of venom injection. In addition provide clear evidence of the involvement of the KKS in inflammatory response induced by S. plumieri venom.

Biochemical characterization of a cysteine proteinase from Bauhinia forficata leaves and its kininogenase activity

In this work, the proteinase activity detect in the acetone precipitate (80%, v/v) of B. forficata leaves, trivially known as cow paw, and popularly used in folk medicine for treatment of diabetes mellitus, was purified by chromatography on Sephadex G-25, Canecystatin-Sepharose, and on Con A-Sepharose. The molecular weight 30kDa was estimated by SDS-PAGE and zymography, and the N-terminal sequence and CD spectra indicated a relationship with the papain family of cysteine proteinases. Denominated baupain, the enzyme was activated by dithiotreitol and inhibited by E-64 and iodoacetamide, but not by benzamidine, TLCK, TPCK and EDTA. The S2 and S1 substrate specificity of baupain, assayed with two series of fluorescence resonance energy transfer (FRET) peptide substrates derived from Abz-KLRSSK-Q-EDDnp, indicates a preference for Phe and Tyr at P2 position over Leu found in papain. Baupain releases bradykinin from HMWK (human high molecular weight kininogen) though its proteolytic activity is blocked by the sequence motif QVVA of kininogen (Kiapp=1.9×10−8M). Canecystatin, from sugar cane, which also lodges the QVVA sequence, inhibits baupain (Kiapp=0.18×10−9M).

Substrate specificity and inhibition of human kallikrein-related peptidase 3 (KLK3 or PSA) activated with sodium citrate and glycosaminoglycans

We report the enzymatic properties and substrate specificity of human recombinant KLK3 in the presence of glycosaminoglycans (GAGs) and sodium citrate. This salt is highly concentrated in prostate and in its presence KLK3 had a similar hydrolytic efficiency as chymotrypsin. In contrast to the latter peptidase, KLK3 activated by sodium citrate efficiently hydrolyzed substrates containing R, H and P at the P1 position. Activated KLK3 also cleaved peptides derived from the bradykinin domain of human kininogen at the same sites as human kallikrein KLK1, but presented low kininogenase activity. Angiotensin I has several sites for hydrolysis by KLK3; however, it was cleaved only at the Y–I bond (DRVY↓IHPFHL). Sodium citrate modulated KLK3 conformation as observed by alterations to the intrinsic fluorescence of phenylalanines and tryptophans. Activated KLK3 was reversibly inhibited by Z-Pro-Prolinal and competitively inhibited by ortho-phenantroline. Together, these are noteworthy observations for the future design of specific non-peptide inhibitors of KLK3 and to find natural substrates.

Using kallikrein-related peptidases (KLK) as novel cancer biomarkers

Thromb Haemost 2009; 101: 222–224 The scientist Emil-Karl Frey, a scholar of the famous surgeon ‘Geheimrat’ Ferdinand Sauerbruch, observed in 1925 a considerable reduction in arterial blood pressure when he injected human urine into dogs, an until then unknown cardioactive and vasoactive effect which he attributed to an unspecified substance with potential biological functions (1, 2). This then uncharacterized kininogenase was named kallikrein (Greek synonym for pancreas: kallikreas) by the three German scientists H. Kraut, E.-K. Frey and E. Werle, who in 1930 reported that the pancreas is a rich source of this endogenous hypotensive substance (3). A few years later, E.-K. Werle identified kallikrein as a proteolytic enzyme (KLK1 [EC 3.4.21.35] initially named tissue kallikrein, glandular kallikrein, kallikrein 1 or hk1) that liberates the biologically highly active, basic polypeptide ‘DK’ or kallidin (i.e. lys-bradykinin) from the blood plasma protein kallidinogen (now termed highand low-molecular-weight kininogen, HMWKand LMWK-kininogen) (4). The serine protease KLK1 is different from the later discovered plasma serine protease kallikrein (KLKB1 or Fletcher factor [EC 3.4.21.34], located on chromosome 4q34-q35) which is part of the plasma contact activation system and structurally related to coagulation factor XI (5), whereas KLK1 located on chromosome 19q13.4 is structurally related to the serine protease trypsin (5–7). Later on, two more glandular kallikreins were identified in humans: KLK2 (initially named human glandular kallikrein 1, hGK1, and later human kallikrein 2, hK2) (8) and KLK3 (also known as prostate-specific antigen [PSA]) (9, 10), both located on chromosome 19q13.4 as well (6). Some time later, before the turn of the millenium, 12 additional serine protease genes were independently discovered by three major research groups to be tandemly located on chromosome 19q13.4 and therefore assigned to the glandular kallikrein family as well, based on their localization to chromosome locus 19q13.4 and sequence/structural similarities with the three traditional glandular kallikrein genes (11–13), thus forming a new, “extended” kallikrein gene family (6, 7). Hence, a new comprehensive nomenclature was needed, and since 2006 of the 15 tissue-related kallikreins 14 are denoted kallikrein-related peptidases (KLK2 to KLK15) (14), except for kallikrein 1, since this is the only serine protease in the KLK-family with significant kininogenase activity (15). Now, with the comprehensive description of the human KLK gene locus available, one of the main bio-medical research endeavors is focused on the clarification of the (patho)physiological functions of the KLK (16). KLK are expressed in a wide range of tissues, although individual KLK do have quite restricted expression patterns suggesting a functional role in diverse (patho)physiological processes (17), e.g. skin desquamation and other skin diseases, tooth development and enamel defects, Alzheimer’s disease, Parkinson’s disease, and cancer (6, 15). Besides KLK3 (PSA), a well known biomarker for prostate cancer, other members of the KLK family are now considered as potential biomarkers in malignant disease states as well. Recently, several of the kallikrein-related peptidases have shown promise as prognostic and predictive cancer biomarkers, e.g. for cancer of the prostate, testis, kidney, breast, ovary, lung, colon, pancreas, and brain (6, 18). Interestingly, some of the KLK can auto-activate, while others activate each other, suggesting that the KLK may be part of an enzymatic cascade similar to the urokinase/plasminogen system or the matrix metalloprotease family, which are associated with the malignant potential of multiple malignant diseases (19, 20). Currently, three well-established technologies are in use to quantify kallikrein-related peptidase expression: RT-PCR (mRNA), ELISA (protein) and DNA-methylation (epigenetic modification of CpG islands in genomic DNA) (6, 21). From these assays, it can be assumed that certain members of the kallikrein-related peptidase gene family are differentially expressed in a wide variety of carcinomas. Although still under investigation, it has been shown that these factors can serve as new biomarkers for diagnosis, prognosis, and monitoring of cancer (6, 7, 15, 16). In Figure 1A the number of citations per year are displayed with reference to KLK3 (PSA) and cancer, encompassing investigations in human and animal tissues and cell lines, respectively. Between 1982 and 2008, 12,135 articles were published in MedLine including 1,631 reviews. Most of the experiments were conducted with human tumor tissues, tumor cell lines or blood components (12,047); 1,523 considering clinical trials. Only a small portion of the published articles dealing with the clinical impact of kallikrein-related peptidases (556, includ© 2009 Schattauer GmbH, Stuttgart

SUPPRESSION OF MAFS EXPRESSION BY THE SIRNA TECHNIQUE ALTERS THE GENE PROFILE RELATED TO PANCREATIC ENDOCRINE HORMONE AND ADIPOCYTOKINE IN VIVO AND IN VITRO

One of the large maf molecules, mafA, is a strong transactivator of insulin, and other mafs, including mafB and c-maf, have been found to be involved in the regulation of pancreatic development and function. In a previous study we established an mRNA interference technique that makes it possible to modify the level of targeted mRNAs in the mouse in vivo. An expression vector carrying synthetic maf-siRNA was rapidly injected into the tail vein of 8-week-old-mice (Hydrodynamic method), and the resulting alteration of the gene profile was analyzed with a microarray system. Suppression of the mafA mRNA level in the pancreas by siRNA resulted in down-regulation of insulin and glucagon mRNA and of the mRNA of the adipocytokines adipsin and adiponectin. Thus, mafA is likely to be involved in the regulation of adipocytokines as well as pancreatic endocrine hormone. In a preliminary experiment we found that transfection of adipocytes with mafA siRNA in vitro attenuated adipocyte differentiation and downregulated adipokine gene expression. On the other hand, the c-maf expression level in the pancreas of c-maf-siRNA-treated mice was approximately 50% reduced in comparison with stop-siRNA-treated animals. Microarray analysis revealed down-regulation of expression of the insulin and glucagon genes in the pancreas of siRNA-treated mice, while Ddit3 and Nurp1 (Ddit3 having a p53 binding site, encodes an inhibitor of C/EBP and Nurp1 is a transcriptional cofactor activated in acinar cells of pancreatitis) were upregulated, and HSP8 expression was down-regulated. Moreover, several genes coding kallikrein 1-related peptidase (Klk1b4, Klk1b8, Klk1b11 and Klk1b24), that locate on kallikrein 1 locus (chr.19q13.3-13.4) and code serin protease protein without proven kininogenase activity were down-regulated in the pancreas. This study demonstrated that reduction of mafA and c-maf RNA levels altered expression of genes that code pancreatic hormones, including insulin and glucagon. The results of this study also suggest that mafA may regulate several adipocytokines in the pancreas and that c-maf may not only regulate endocrine hormones in the pancreas but serine proteases as well. Taken together, the findings in this study suggested there the involvement of a network of transcriptional factors, mafs, pancreatic endocrine hormones, and adipocytokines in glucose and lipid metabolism.

Contribution of Coagulation Pathways and Fibrinolysis in Generation of Kinins.

BACKGROUND. Classically, there are two main pathways by which kinins are generated: the plasma and tissue kallikrein-kinin systems. Each of this complex multi-protein system includes tissue or plasma kallikrein, which generate kinins from high or low molecular weight kininogen. In addition to plasma and tissue kallikreins, other serine proteases may have a kinin-forming capacity.OBJECTIVES. The main purpose of this study was to characterize the kininogenase activity of plasma kallikrein, thrombin (factor IIa), factor Xa, and plasmin in a purified system. We also aimed at characterizing the kinetic profile of generation of kinins in a whole blood system where coagulation and fibrinolysis were monitored by thromboelastography.METHODS. Bradykinin (BK) released from both low and high molecular weight kininogens and quantified with a specific competitive enzyme immunoassay was used to calculate the kinetic parameters for the different kininogenase activities. Physicochemical and pharmacological characterization of immunoreactive kinins confirm their nature. The kinetic profile of kinin generation was studied during the process of coagulation which was initiated with kaolin or with tissue factor and monitored by thromboelastography.RESULTS. BK was released not only by plasma kallikrein; both plasmin and factor Xa hydrolyzed high molecular kininogen to produce BK. In the case of thrombin, the release of both BK and kallidin was detected from low and high molecular weight kininogen. When applied to whole blood, this analytical approach indicated that the kinin-forming capacity of the coagulation intrinsic pathway was higher than that of the extrinsic pathway. The triggering of fibrinolysis by tissue plasminogen activator dramatically potentiated the release of des-Arg9-BK.CONCLUSIONS. Although plasma kallikrein is considered as being the main kinin-forming enzyme, other serine proteases of the coagulation and the fibrinolysis pathways are able to generate kinins and could play a role in different pathologies where kinins are potentially involved.

Modulation of hypotensive effects of kinins by cathepsin K

Kinins are pro-inflammatory peptides, which participate in the maintenance of cardiovascular homeostasis, and play a key role in numerous diseases, including lung fibrosis and hypertension. Evidence has been provided recently for the presence of alternative mechanisms of bradykinin generation and/or degradation. Here we showed that cathepsin K may act as a potent kinin-degrading enzyme in bloodstream. Contrary to cathepsin L, cathepsin K attenuates kallikrein-induced decrease of rat blood pressure, and reduces the hypotensive effect of bradykinin in a dose-dependant manner. Moreover, we identified, by engineering the S2 subsite of both recombinant enzymes, two critical residues involved respectively in the kininase activity of cathepsin K, i.e. Tyr67/Leu205, versus kininogenase activity of cathepsin L, i.e. Leu67/Ala205. In conclusion, according to its ability to modulate hypotensive effects of kinins, we propose that cathepsin K is a kininase of biological relevance, in complement of well-documented neutral endopeptidase or angiotensin-converting enzyme.

Transcriptome analysis of expressed sequence tags from the venom glands of the fish Thalassophryne nattereri

Thalassophryne nattereri (niquim) is a venomous fish found on the northern and northeastern coasts of Brazil. Every year, hundreds of humans are affected by the poison, which causes excruciating local pain, edema, and necrosis, and can lead to permanent disabilities. In experimental models, T. nattereri venom induces edema and nociception, which are correlated to human symptoms and dependent on venom kininogenase activity; myotoxicity; impairment of blood flow; platelet lysis and cytotoxicity on endothelial cells. These effects were observed with minute amounts of venom. To characterize the primary structure of T. nattereri venom toxins, a list of transcripts within the venom gland was made using the expressed sequence tag (EST) strategy. Here we report the analysis of 775 ESTs that were obtained from a directional cDNA library of T. nattereri venom gland. Of these ESTs, 527 (68%) were related to sequences previously described. These were categorized into 10 groups according to their biological functions. Sequences involved in gene and protein expression accounted for 14.3% of the ESTs, reflecting the important role of protein synthesis in this gland. Other groups included proteins engaged in the assembly of disulfide bonds (0.5%), chaperones involved in the folding of nascent proteins (1.4%), and sequences related to clusterin (1.5%), as well as transcripts related to calcium binding proteins (1.0%). We detected a large cluster (1.3%) related to cocaine- and amphetamine-regulated transcript (CART), a peptide involved in the regulation of food intake. Surprisingly, several retrotransposon-like sequences (1.0%) were found in the library. It may be that their presence accounts for some of the variation in venom toxins. The toxin category (18.8%) included natterins (18%), which are a new group of kininogenases recently described by our group, and a group of C-type lectins (0.8%). In addition, a considerable number of sequences (32%) was not related to sequences in the databases, which indicates that a great number of new toxins and proteins are still to be discovered from this fish venom gland.