Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

Protease activity in Giardia duodenalis trophozoites of axenic strains isolated from symptomatic and asymptomatic patients

We have examined by gelatin-SDS-PAGE the protease activity in cell lysates of Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic patient (BTU-11) and an asymptomatic carrier (BTU-10), and the reference strain Portland 1 (P1). The proteolysis band patterns showed differences among strains isolated from asymptomatic and symptomatic individuals. The lysate of the strain BTU-10, showed only five hydrolysis bands, while a greater number of bands (10-11 bands) was seen in strains BTU-11 and P1. The protease activity in all lysates was inhibited by cysteine (E-64 and iodoacetamide) and serine proteases (TPCK and TLCK) inhibitors, but not by PMSF and EDTA. In general, the results revealed protease activities in G. duodenalis trophozoites of Brazilian axenic strains and the predominance of cysteine proteinases. It should be stressed the inter-strain difference in hydrolysis band patterns observed between strains isolated from symptomatic patients and the strain obtained from an asymptomatic carrier.

Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 Proteolytic activity is catalytically related to the HslVU protease?

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (β-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P 1, although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli Hs1VU protease suggested two putative threonine catalytic groups, one in the N-domain (T 136, K 168, and Y 264) and the other in the C-domain (T 375, K 409, and S 502). Mutagenesis studies showed that the replacement of K 409 by A caused a complete loss of the proteolytic activity, whereas the mutation of K 168 to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T 375, K 409, and S 502 at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Increased pancreatic islet mass is accompanied by activation of the insulin receptor substrate-2/serine-threonine kinase pathway and augmented cyclin D(2) protein levels in insulin-resistant rats

It is well known that glucocorticoids induce peripheral insulin resistance in rodents and humans. Here, we investigated the structural and ultrastructural modifications, as well as the proteins involved in beta-cell function and proliferation, in islets from insulin-resistant rats. Adult male Wistar rats were made insulin resistant by daily administration of dexamethasone (DEX; 1mg/kg, i.p.) for five consecutive days, whilst control (CTL) rats received saline alone. Structure analyses showed a marked hypertrophy of DEX islets with an increase of 1.7-fold in islet mass and of 1.6-fold in islet density compared with CTL islets (P < 0.05). Ultrastructural evaluation of islets revealed an increased amount of secreting organelles, such as endoplasmic reticulum and Golgi apparatus in DEX islets. Mitotic figures were observed in DEX islets at structural and ultrastructural levels. Beta-cell proliferation, evaluated at the immunohistochemical level using anti-PCNA (proliferating cell nuclear antigen), showed an increase in pancreatic beta-cell proliferation of 6.4-fold in DEX islets compared with CTL islets (P < 0.0001). Increases in insulin receptor substrate-2 (IRS-2), phosphorylated-serine-threonine kinase AKT (p-AKT), cyclin D(2) and a decrease in retinoblastoma protein (pRb) levels were observed in DEX islets compared with CTL islets (P < 0.05). Therefore, during the development of insulin resistance, the endocrine pancreas adapts itself increasing beta-cell mass and proliferation, resulting in an amelioration of the functions. The potential mechanisms that underlie these events involve the activation of the IRS-2/AKT pathway and activation of the cell cycle, mediated by cyclin D(2). These adaptations permit the maintenance of glycaemia at near-physiological ranges.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Inserções de aminoácidos na protease têm sido raramente descritas em pacientes infectados pelo HIV. Uma destas inserções foi, recentemente, descrita no codon 35, embora seu impacto na resistência mantém-se pouco conhecido. Este trabalho apresenta um caso de uma variante viral com inserção no codon 35 da protease, descrita pela primeira vez em Bauru, São Paulo, Brasil, circulante em um homem, caucasiano, com 38 anos, o qual apresenta infecção assintomática pelo HIV desde 1997. A variante isolada mostrou uma inserção no codon 35 da protease de dois aminoácidos: uma treonina e um ácido aspártico, resultando na sequência de aminoácidos E35E_TD.

Crystallization, preliminary X-ray analysis and Patterson search of a new aspartic protease isolated from human urine

Aspartic protease (EC 3.4.23) make up a widely distributed class of enzymes in animals, plants, microbes and, viruses. In animals these enzymes perform diverse functions, which range from digestion of food proteins to very specific regulatory roles. In contrast the information about the well-characterized aspartic proteases, very little is known about the corresponding enzyme in urine. A new aspartic protease isolated from human urine has been crystallized and X-ray diffraction data collected to 2.45 Angstrom resolution using a synchrotron radiation source. Crystals belong to the space group P2(1)2(1)2(1) the cell parameters obtained were a=50.99, b=75.56 and c=89.90 Angstrom. Preliminary analysis revealed the presence of one molecule in the asymmetric unit. The structure was determined using the molecular replacement technique and is currently being refined using simulated annealing and conjugate gradient protocols.

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.

Characterisation of protease activity in extracellular products secreted by Giardia duodenalis trophozoites treated with propolis

Results from our laboratory revealed propolis activity on Giardia trophozoites proliferation. Since therapeutic agents can inhibit the activity of proteases related to relevant biologic and physiologic processes of parasites, this study was undertaken to characterise the proteolytic activity of excretory/secretory products (ESP) of trophozoites treated with propolis. ESP was obtained from culture supernatants of trophozoites exposed to 250 and 500 mu g mL(-1) of propolis. ESP were tested in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the protein profiles and the protease activity was assayed in gelatin-containing gels. Synthetic inhibitors were used to characterise the protease classes. Treated and non-treated ESP showed a similar protein and hydrolysis pattern. A simple pattern of protein composed by five evident bands of approximately 167, 132, 79, 61 and 51 kDa was found, and the zymograms comprised hydrolysis zones distributed from > 170 to 23 kDa. No inhibition was seen on protease activity of propolis-treated trophozoites, whose hydrolysis pattern was similar to control. One may conclude that both ESP degraded gelatin and the activity was predominantly due to cysteine proteases. Although propolis had no effect on the proteolytic activity, further studies could identify the active constituents responsible for propolis antigiardial activity and their mechanisms of action.

Mucosa-associated but not luminal Escherichia coli is augmented in Crohn's disease and ulcerative colitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Giardia duodenalis: protein substrates degradation by trophozoite proteases

The present investigation was undertaken to identify and characterize trophozoite proteases of five axenic strains of Giardia duodenalis isolated in Brazil and the reference strain Portland 1 isolated in the United States. Trophozoite cell lysates of each strain were analysed for the pattern of proteins and for proteolytic activity. Samples were tested in SDS-polyacrylamide gel electrophoresis for the protein profiles, and the detection of proteases in cell lysates was performed using substrate gel electrophoresis [gelatin, collagen, bovine serum albumin (BSA) and haemoglobin] and azocasein assays. Indeed, synthetic inhibitors were included in the assays to characterize the protease classes. Differences on the hydrolysis patterns of protein substrates were observed in relation to the substrate composition as much as the Giardia trophozoite strain. The substrate-containing gels revealed hydrolysis bands with molecular masses ranging from > 97 to 20-15 kDa, and most zones were common to the five strains. However, some pronounced differences could be detected in the BTU-11 pattern. Azocasein was also degraded; however, depending on the lysate assayed, the degree of substrate degradation was variable. It was observed that inhibitory effects are substrate-dependent since the activity was predominantly due to cysteine proteases against gelatin, collagen, BSA and azocasein substrates and due to serine against haemoglobin. The presence of aspartic protease and aminopeptidase activity in the lysates was also indicated.

Identification and characterization of excretory/secretory products of Giardia duodenalis trophozoites

One of the major questions concerning Giardia is the understanding of pathophysiological processes associated with small intestine abnormalities. There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in the host intestinal epithelium. The present investigation was undertaken to examine the protease activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). E/S products from trophozoites of each strain in conditioned medium were tested with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for the protein profiles, and the protease activity was analyzed using substrate-impregnated SDS-PAGE (gelatin and collagen) and hemoglobin assay. The proteases characterization was based on inhibition assays including synthetic inhibitors. Electrophoresis analysis of E/S products revealed a banding pattern composed by few bands (4 to 6 bands) in the migration region of 123 to 28 kDa. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted substrate degradation and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibitor assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases, although the presence of serine proteases was also indicated. Degradation of substrates including collagen and hemoglobin could lead us to speculate different functions of Giardia excreted/secreted proteases in vivo, but to confirm this possibility and to elucidate its implication on host-parasite interactions, further experiments applying protocols for the purification of proteases are necessary. Even so, our observations are relevant and hold the perspective for the understanding about protease activity in Giardia trophozoites of axenic strain isolated in an endemic area.

Characterization of the excretory/secretory products of Dermatobia hominis larvae, the human bot fly

Proteolytic activity in excretory/secretory products (ESP) of first- (L1), second- (L2) and third-instar (L3) larvae of Dermatobia hominis was analyzed through gelatin-gel and colorimetric enzyme assays with the chromogenic substrates azocasein and BApNA. The functional characterization of proteases was based on inhibition assays including synthetic inhibitors. ESP were obtained from new-hatched larvae reared in the laboratory and from second- and third-instar larvae removed from naturally infested cattle. Gelatin-gel analysis evidenced few bands of proteolysis, predominantly of high apparent molecular masses, in ESP of L1, whereas in the gel of L2 and U ESP there was a wide range of proteolytic activity most of them not resolved in a single species. Azocasein assays revealed a progressive increase of protease activity from first- to third-instar larvae. Protease inhibitor assays revealed a predominance of metalloproteases in L1 ESP that could be related to a skin penetration process and to a diversion of host immune response. The predominance of serine proteases in L2 and L3 and the great tryptic activity presented by L3 ESP were attributed to an increasing trophic activity by the growing larvae, since the viability of adult flies strictly depends on larval abilities to assimilate nutrients from the host. Taking together, these results suggest that Dematobia larvae secrete/excrete different proteases that may be related to diverse functions during host penetration and infestation, which reinforces the relevance of the study of such proteolytic enzymes. (C) 2009 Elsevier B.V. All rights reserved.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.