Guacyara Motta - Academia.edu (original) (raw)

Papers by Guacyara Motta

Frontiers in Physiology, Jul 11, 2017

Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In ... more Human plasma kallikrein-kinin system proteins are related to inflammation through bradykinin. In the proximity of its target cells, high molecular weight kininogen (H-kininogen) is the substrate of plasma kallikrein, which releases bradykinin from H-kininogen. Heparan sulfate proteoglycans (HSPGs) play a critical role in either recruiting kinin precursors from the plasma, or in the assembly of kallikrein-kinin system components on the cell surface. Furthermore, HSPGs mediate the endocytosis and activation of H-kininogen and plasma prekallikrein. In the presence of HSPGs (Chinese hamster ovary cell, CHO-K1, wild type cells) both heparin and heparan sulfate strongly inhibit the H-kininogen interaction with the cell membrane. H-kininogen is internalized in endosomal acidic vesicles in CHO-K1 but not in CHO-745 cells (mutant cells deficient in glycosaminoglycan biosynthesis). The endocytosis process is lipid raft-mediated and is dependent on caveolae. Both types of CHO cells do not internalize bradykinin-free H-kininogen. At pH 7.35, bradykinin is released from H-kininogen on the surface of CHO-745 cells only by serine proteases; however, in CHO-K1 cells either serine or cysteine proteases are found to be involved. The CHO-K1 cell lysate contains different kininogenases. Plasma prekallikrein endocytosis in CHO-K1 cells is independent of H-kininogen, and also prekallikrein is not internalized by CHO-745 cells. Plasma prekallikrein cleavage/activation is independent of glycosaminoglycans but plasma kallikrein formation is more specific on H-kininogen assembled on the cell surface through glycosaminoglycans. In this mini-review, the importance of HSPGs in the regulation of plasma kallikrein-kinin system proteins is shown.

PLOS ONE, Mar 30, 2015

Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with ... more Human H-kininogen (120 kDa) plays a role in many pathophysiological processes and interacts with the cell surface through protein receptors and proteoglycans, which mediate Hkininogen endocytosis. In the present work we demonstrate that H-kininogen containing bradykinin domain is internalized and different endogenous kininogenases are present in CHO-K1 cells. We used CHO-K1 (wild type) and CHO-745 (mutant deficient in proteoglycans biosynthesis) cell lines. H-kininogen endocytosis was studied using confocal microscopy, and its hydrolysis by cell lysate fraction was determined by immunoblotting. Bradykinin release was also measured by radioimmunoassay. H-kininogen interaction with the cell surface of CHO-745 cells resulted in bradykinin release by serine proteases. In CHO-K1 cells, which produce heparan and chondroitin sulfate proteoglycans, internalization of H-kininogen through its bradykinin domain can occur on lipid raft domains/caveolae. Nevertheless bradykinin-free H-kininogen was not internalized by CHO-K1 cells. The Hkininogen present in acidic endosomal vesicles in CHO-K1 was approximately 10-fold higher than the levels in CHO-745. CHO-K1 lysate fractions were assayed at pH 5.5 and intact H-kininogen was totally hydrolyzed into a 62 kDa fragment. By contrast, at an assay pH 7.4, the remained fragments were 115 kDa, 83 kDa, 62 kDa and 48 kDa in size. The antipain-Sepharose chromatography separated endogenous kininogenases from CHO-K1 lysate fraction. No difference was detected in the assays at pH 5.5 or 7.4, but the proteins in the fraction bound to the resin released bradykinin from H-kininogen. However, the proteins in the unbound fraction cleaved intact H-kininogen at other sites but did not release bradykinin. H-kininogen can interact with extravascular cells, and is internalized dependent on its bradykinin domain and cell surface proteoglycans. After internalization, H-kininogen is proteolytically processed by intracellular kininogenases. The present data also demonstrates that serine or cysteine proteases in lipid raft domains/caveolae on the CHO cell can hydrolyze H-kininogen, thus releasing kinins.

Blood, Jan 15, 1998

The consequences of assembling the contact system of proteins on the surface of vascular cells ha... more The consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent K d of 23 ؎ 11 nmol/L, B max of 1.7 ؎ 0.5 ؋ 10 7 sites per cell [mean ؎ SD, n ‫؍‬ 5 experiments]). Cellassociated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK•PK complexes is entirely independent of exogenous factor XII (K m ‫؍‬ 30 nmol/L, V max ‫؍‬ 12 ؎ 3 pmol/L/min in the absence v K m ‫؍‬ 20 nmol/L, V max ‫؍‬ 9.2 ؎ 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cellassociated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, releasing kallikrein from the surface. Furthermore, activation of PK bound to HK on HUVECs promotes kallikrein-dependent activation of pro-urokinase, resulting in the formation of plasmin. These results indicate the existence of a previously undescribed, factor XII-independent pathway for contact factor activation on HUVECs that regulates the production of bradykinin and may contribute to cell-associated plasminogen activation in vivo. 1998 by The American Society of Hematology. MATERIALS AND METHODS Proteins. HK was purified from plasma using sequential carboxymethyl-papain-Sepharose (CM-papain-Sepharose) and Blue-Sepharose affinity chromatography as previously reported. 28,29 HK migrated as a 120-kD protein on sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis (SDS-PAGE) after reduction with 2% ␤-mercaptoethanol. HK had a specific activity of 12 to 20 U/mg. Purified HK was iodinated with IODOGEN (Pierce, Rockford, IL) as previously reported. 20 Human PK was purchased from Enzyme Research Laboratories (South Bend, IN). The protein migrated as a doublet at 88 and 85 kD on 10% SDS-PAGE under reduced conditions and expressed approximately 1% to 3% of the amidolytic activity of kallikrein. 33 No FXII or its activated forms were found in the HK or PK preparations by immunoblotting using a monospecific goat antisera to human FXII. PK was also iodinated with IODOGEN using identical techniques previously reported for HK. 20,23 Iodinated PK was a doublet at 88 and 85 kD on 10% SDS-PAGE under reducing conditions. FXII, purchased from Enzyme Research Laboratories, migrated predominantly as a single band at 80 kD on 10% SDS-PAGE under reduced conditions and expressed less than 1% of the amidolytic activity of activated FXII. Activated factor XII (␣FXIIa) was purchased from Enzyme Research Laboratories. ␣FXIIa migrated as two bands at 50 and 28 kD on 10%

Journal of Neurochemistry, Jul 10, 2019

Temporal lobe epilepsy (TLE) is a chronic disease, characterized by severe and refractory seizure... more Temporal lobe epilepsy (TLE) is a chronic disease, characterized by severe and refractory seizures, triggered in the hippocampus and/or amygdala, disrupting the blood brain barrier (BBB). This disruption can sustain, or aggravate, the epileptic condition. The aim of this study was to evaluate the activation of the kallikrein-kinin system (KKS) in patients with TLE, as it relates to maintenance of the BBB. Human hippocampal sclerotic tissues, removed after surgery for seizure control, plasma, and serum were used in the following assays: immunostaining for white blood cells in the TLE hippocampus, C-reactive protein in serum, quantification of plasma kallikrein (PKal) and cathepsin B (CatB) activity in serum and plasma, quantification of C1-inhibitor, analysis of high-molecular-weight kininogen (Hkininogen) fragments, and activation of plasma prekallikrein for comparison with healthy controls. Infiltration of white blood cells in the sclerotic hippocampus, and a significant increase in the neutrophil/lymphocyte ratio in the blood of TLE patients were observed. High levels of C-reactive protein (TLE = 1.4 ± 0.3 µg/mL), PKal (TLE = 5.4 ± 0.4 U/mL) and CatB (TLE = 4.9 ± 0.4 U/mL) were also evident in the serum of TLE patients comparing to controls. A strong linear correlation was observed between active CatB and PKal in the serum of TLE patients (r = 0.88). High levels of cleaved H-kininogen and free PKal, and low levels of C1-inhibitor (TLE = 188 ± 12 µg/mL) were observed in the serum of TLE patients. Our data demonstrated that the plasma KKS is activated in patients with TLE.

Microorganisms, Sep 1, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

Biochimie, Oct 1, 2011

Dedicated to the memory of Claudio A.M. Sampaio and Carl Peter von Dietrich for their contributio... more Dedicated to the memory of Claudio A.M. Sampaio and Carl Peter von Dietrich for their contributions to the fields of proteases and glycosaminoglycans.

Biological Chemistry, Nov 29, 2008

In this study, we analyzed the influence of proteoglycans on the interaction between human high m... more In this study, we analyzed the influence of proteoglycans on the interaction between human high molecular weight kininogen (HK) and the cell surface. We found that D5-related peptide inhibits HK-biotin cellular uptake. Confocal microscopy showed that HK colocalizes with heparan sulfate proteoglycan (HSPG) at the cell surface. When biotin-HK is incubated with rabbit aorta endothelial cells (RAECs) and CHO-K1 cells, it is internalized into acidic intracellular vesicles, whereas when incubated with CHO-745 cells, which express reduced levels of glycosaminoglycans, HK is not internalized. To further verify the hypothesis that HSPG-dependent mechanisms are involved in HK uptake and proteolytic processing in lysosomes, we tested chloroquine, which blocks Alexa 488-HK colocalization with Lyso Tracker in acidic endosomal vesicles. The process of HK internalization was blocked by low temperatures, methyl-β-cyclodextrin, FCCP and 2-deoxy-d-glucose, implying that HK uptake into acidic vesicles is energy-dependent and most likely involves binding to HSPG structures localized in cholesterol-rich domains present in the plasma membrane. Kinin generation at the cell surface was much higher in tumorigenic cells (CHO-K1) when compared to endothelial cells (RAECs). The present data indicate that the process of HK endocytosis involving HSPG is a novel additional mechanism which may control kinin generation at the cell surface.

Thrombosis and Haemostasis, 2001

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK ... more Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires &amp;amp;amp;gt; or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.

FEBS Letters, Jun 26, 2002

Investigations determined that the cell matrix-associated prekallikrein (PK) activator is prolylc... more Investigations determined that the cell matrix-associated prekallikrein (PK) activator is prolylcarboxypeptidase. PK activation on human umbilical vein endothelial cell (HUVEC) matrix is inhibited by antipain (IC 50 = 50 W WM) but not antifactor XIIa antibody, 3 mM benzamidine, 5 mM iodoacetic acid or iodoacetamide, or 3 mM N-ethylmaleimide. Corn trypsin inhibitor (IC 50 = 100 nM) or Fmoc-aminoacylpyrrolidine-2nitrile (IC 50 = 100 W WM) blocks matrix-associated PK activation. Angiotensin II (IC 50 = 100 W WM) or bradykinin (IC 50 = 3 mM), but not angiotensin 1^7 or bradykinin 1^5, inhibits matrix-associated PK activation. ECV304 cell matrix PK activator also is blocked by 100 W WM angiotensin II, 1 W WM corn trypsin inhibitor, and 50 W WM antipain, but not angiotensin 1^7. 1 mM angiotensin II or 300 W WM Fmoc-aminoacylpyrrolidine-2-nitrile indirectly blocks plasminogen activation by inhibiting kallikrein formation for single chain urokinase activation. On immunoblot, prolylcarboxypeptidase antigen is associated with HUVEC matrix. These studies indicate that prolylcarboxypeptidase is the matrix PK activator.

Vide Leaf, Hyderabad eBooks, 2020

Veterinary Microbiology, May 1, 2013

Journal of membrane science & technology, 2021

This paper reports the use of camera-less terahertz imaging technique to measure zymogen factor X... more This paper reports the use of camera-less terahertz imaging technique to measure zymogen factor XII (FXII) conformational changes in solution and upon binding to stratum corneum in the absence of its known endogenous potentiating complex, high molecular weight kininogen (HK)-Prekallikrein (PK) in real-time. FXII is synthesized in the liver and secreted into the circulation to serve as a blood coagulation factor. FXII has diverse biological functions. First, it serves as an accessary molecule to extend the generation of thrombin-induced platelet adhesion to the initially formed platelet monolayer. Secondly, FXII provides a robust stimulus for Bradykinin (BK)-induced hyper-permeability by disrupting vascular barriers. Using in vitro assays, studies have shown that FXII activation can occur on negatively charged surfaces. This activation is accelerated in the presence of kallikrein or activated Prekallikrein (PK). We demonstrate our method by measuring the conformational changes that occur upon FXII binding on stratum corneum. The subject of FXII activation remains a work in progress. Previously, Terahertz Scanning Reflectometry (TSR) and Terahertz Spectrometry (TS) was used to demonstrate that different spectral density deviations of the signal are due to the structural alterations and activation of FXII induced by the hydrated stratum corneum. The present work uses Terahertz (THz) imaging to demonstrate that a subset of zymogen FXII bond to the stratum corneum. FXII was not significantly accumulated on the hydrated stratum corneum. However, formed activated FXII (FXIIa) was observed on the stratum corneum. This finding suggests that negatively charged surface is not a necessary condition.

PubMed, 1992

Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West ... more Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, Factor XIIa and human plasma kallikrein. The inhibitors were purified from Enterolobium contortisiliquum (Mr = 23,000), Torresea cearensis (Mr = 13,000), Bauhinia bauhinioides (Mr = 20,000), Bauhinia mollis (Mr = 20,000) and Bauhinia pentandra (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein. B. bauhinioides and B. pentrandra inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa, and B. mollis inhibitor causes trypsin inactivation only. Calculated Ki values were between 10(-7) and 10(-9) M. Chymotrypsin, like trypsin, is also inhibited, but with lower affinity. The trypsin inhibitors, isolated from E. contortisiliquum, B. pentandra, B. bauhinioides and B. mollis seem to be of the Kunitz type; the inhibitor purified from T. cearensis is of the Bowman-Birk type.

Biochimica Et Biophysica Acta - Proteins And Proteomics, Mar 1, 2014

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular... more 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 S 2-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 S 2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S 1 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.

British Journal of Pharmacology, Jun 17, 2010

The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is ... more The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (Kiapp 5.3 nM) and cathepsin G (Kiapp 160.0 nM), and the cysteine peptidase cathepsin L (Kiapp 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by ELISA. KEY RESULTS Pretreatment of the animals with BbCI (2.5 mg•kg-1), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1b or tumour necrosis factor-a, however, did not change. CONCLUSIONS AND IMPLICATIONS Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles. Abbreviations BbCI, Bauhinia bauhinioides serine and cysteine peptidase inhibitor; CINC-1, cytokine-induced neutrophil chemo-attractant-1; IL-1b, interleukin-1beta; Kiapp, apparent inhibitory constant; TNF-a, tumour necrosis factor-alpha

Frontiers in Physiology

Human plasma kallikrein (PKa) is obtained by activating its precursor, prekallikrein (PK), histor... more Human plasma kallikrein (PKa) is obtained by activating its precursor, prekallikrein (PK), historically named the Fletcher factor. Human PKa and tissue kallikreins are serine proteases from the same family, having high- and low-molecular weight kininogens (HKs and LKs) as substrates, releasing bradykinin (Bk) and Lys-bradykinin (Lys-Bk), respectively. This review presents a brief history of human PKa with details and recent observations of its evolution among the vertebrate coagulation proteins, including the relations with Factor XI. We explored the role of Factor XII in activating the plasma kallikrein–kinin system (KKS), the mechanism of activity and control in the KKS, and the function of HK on contact activation proteins on cell membranes. The role of human PKa in cell biology regarding the contact system and KSS, particularly the endothelial cells, and neutrophils, in inflammatory processes and infectious diseases, was also approached. We examined the natural plasma protein in...

Biological Chemistry, 2004

Plasma kallikrein plays a role in coagulation, fibrinolysis and inflammation. Cathepsins B and L ... more Plasma kallikrein plays a role in coagulation, fibrinolysis and inflammation. Cathepsins B and L participate in (patho)physiological processes such as peptide antigen processing, tissue remodeling events, protein turnover in cells, hormone processing and tumor invasion. The present work analyzes the processing of prekallikrein/kallikrein by lysosomal cathepsins. Prekallikrein is not hydrolyzed by catB, and catL generates an inactive fragment of prekallikrein. Both kallikrein chains are hydrolyzed by catL and the light chain is mainly hydrolyzed by catB; kallikrein activity is lower after incubation with catL compared to catB. Our data suggest that the plasma kallikrein/kinin system can be controlled by cathepsins.

BV UNIFESP: Teses e dissertaçõe

Microorganisms

We aimed to determine the biomarker performance of the proteolytic enzymes cathepsin B (Cat B) an... more We aimed to determine the biomarker performance of the proteolytic enzymes cathepsin B (Cat B) and plasma kallikrein (PKa) and transforming growth factor (TGF)-β to detect hepatic fibrosis (HF) in chronic hepatitis C (CHC) patients. We studied 53 CHC patients and 71 healthy controls (HCs). Hepatic-disease stage was determined by liver biopsies, aminotransferase:platelet ratio index (APRI) and Fibrosis (FIB)4. Hepatic inflammation and HF in CHC patients were stratified using the METAVIR scoring system. Cat-B and PKa activities were monitored fluorometrically. Serum levels of TGF-β (total and its active form) were determined using ELISA-like fluorometric methods. Increased serum levels of Cat B and PKa were found (p < 0.0001) in CHC patients with clinically significant HF and hepatic inflammation compared with HCs. Levels of total TGF-β (p < 0.0001) and active TGF-β (p < 0.001) were increased in CHC patients compared with HCs. Cat-B levels correlated strongly with PKa levels ...