Homogeneous, Synthetic, Non-Saccharide Glycosaminoglycan Mimetics as Potent Inhibitors of Human Cathepsin G.

Cathepsin G (CatG) is a pro-inflammatory neutrophil serine protease that is important for host defense, and has been implicated in several inflammatory disorders. Hence, inhibition of CatG holds much therapeutic potential; however, only a few inhibitors have been identified to date, and none have reached clinical trials. Of these, heparin is a well-known inhibitor of CatG, but its heterogeneity and bleeding risk reduce its clinical potential. We reasoned that synthetic small mimetics of heparin, labeled as non-saccharide glycosaminoglycan mimetics (NSGMs), would exhibit potent CatG inhibition while being devoid of bleeding risks associated with heparin. Hence, we screened a focused library of 30 NSGMs for CatG inhibition using a chromogenic substrate hydrolysis assay and identified nano- to micro-molar inhibitors with varying levels of efficacy. Of these, a structurally-defined, octasulfated di-quercetin NSGM 25 inhibited CatG with a potency of ~50 nM. NSGM 25 binds to CatG in an allosteric site through an approximately equal contribution of ionic and nonionic forces. Octasulfated 25 exhibits no impact on human plasma clotting, suggesting minimal bleeding risk. Considering that octasulfated 25 also potently inhibits two other pro-inflammatory proteases, human neutrophil elastase and human plasmin, the current results imply the possibility of a multi-pronged anti-inflammatory approach in which these proteases are likely to simultaneously likely combat important conditions, e.g., rheumatoid arthritis, emphysema, or cystic fibrosis, with minimal bleeding risk.

Serpins in Fasciola hepatica: insights into host-parasite interactions.

Protease inhibitors play crucial roles in parasite development and survival, modulating the immune responses of their vertebrate hosts. Members of the serpin family are irreversible inhibitors of serine proteases and regulate systems related to defence against parasites. Limited information is currently available on protease inhibitors from the liver fluke Fasciola hepatica. In this study, we characterised four serpins from F. hepatica (FhS-1-FhS-4). Biochemical characterisation revealed that recombinant FhS-2 (rFhS) inhibits the activity of human neutrophil cathepsin G, while rFhS-4 inhibits the activity of bovine pancreatic chymotrypsin and cathepsin G. Consistent with inhibitor function profiling data, rFhS-4 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner.Similar to other serpins, rFhS2 and rFhS-4 bind to heparin with high affinity. Tissue localisation demonstrated that these serpins have different spatial distributions. FhS-2 is localised in the ovary, while FhS-4 was found in gut cells. Both of them co-localised in the spines within the tegument. These findings provide the basis for study of functional roles of these proteins as part of an immune evasion mechanism in the adult fluke, and in protection of eggs to ensure parasite life cycle continuity. Further understanding of serpins from the liver fluke may lead to the discovery of novel anti-parasitic interventions.

Inhibition of Neutrophil Elastase and Cathepsin G As a New Approach to the Treatment of Psoriasis: From Fundamental Biology to Development of New Target-Specific Drugs.

Psoriasis therapy remains an extremely relevant area of modern drug design, due to necessity of adverse reaction reduction, inherent for actual methods of therapy. It was established that two serine proteases-neutrophil elastase 1 (HNE1) and cathepsin G (CatG)-are the key agents in psoriasis development. The collected molecular data for the presented targets form the basis for the molecular modeling strategy for the search for and identification of new target-specific inhibitors. The result of this work is a group of high-priority small-molecule compounds with double-targeted affinity, which are able to suppress the pro-psoriatic processes induced by the considered serine proteases at the initial stage of the disease.

Cathepsin G Inhibition by Serpinb1 and Serpinb6 Prevents Programmed Necrosis in Neutrophils and Monocytes and Reduces GSDMD-Driven Inflammation.

Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a-/- mice. Furthermore, Sb1a.Sb6a-/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a-/- macrophages showed increased IL-1ß release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD.

Topical treatment with a cathepsin G inhibitor, ß-keto-phosphonic acid, blocks ultraviolet irradiation-induced basement membrane damage in hairless mouse skin.

BACKGROUND: Ultraviolet light (UV) exposure contributes various effects to skin including damage of the basement membrane. Cathepsin G (CTSG) belongs to serine protease family, and its upregulation is involved in wrinkle formation by chronic UV irradiation. However, the effect of CTSG on the basement membrane damage in skin remains unclear. PURPOSE: To investigate the effects of topical treatment with a CTSG inhibitor, ß-keto-phosphonic acid (KPA), on basement membrane damage in chronically UV-irradiated hairless mouse skin. METHODS: The dorsal skin of hairless mice was exposed to UV three times per week for 8 weeks. KPA was applied immediately after each session of UV irradiation. The basement membrane components, CTSG expression, and neutrophil infiltration were analyzed by immunofluorescence staining. The basement membrane structures were visualized by transmission electron microscope. CTSG and MMP-13 protein levels were analyzed by Western blotting. Assessment of wrinkle formation was examined using a skin replica assay. RESULTS: ß-keto-phosphonic acid prevented UV irradiation-induced decrease in type VII collagen, laminin 332, and perlecan at the basement membrane zone and prevented UV-induced breakage of lamina densa and UV-induced shortening of hemidesmosome. KPA prevented UV-induced CTSG and MMP-13 expressions in chronically UV-irradiated hairless mice. Increase in neutrophil infiltration by UV irradiation and UV-induced wrinkle formation was also prevented by KPA. CONCLUSION: Our present study showed the possible involvement of CTSG in UV-induced basement membrane damage in skin through topical treatment with a CTSG inhibitor, KPA. Thus, inhibition of CTSG may be a useful strategy for the prevention of UV-induced basement membrane damage and photoaging.

Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G.

Age-related diseases, such as osteoarthritis, Alzheimer's disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

Inhibition of MMPs Cat G and downregulates the signaling of TGF-ß/Smad in chronic photodamaged human fibroblasts.

OBJECTIVE: To understand the action of Cathepsin G (Cat G) and matrix metalloproteases (MMPs) on the ß/Smad pathway of transforming growth factor ß (TGF-ß) in chronically photodamaged human fibroblasts. Cat G plays a significant role in the process of skin photoaging and in collagen synthesis and degradation which is induced by UV irradiation it could interact with TGF-ß/Smad signaling. No available studies have thoroughly explored its molecular mechanisms of photoaging regulation. PATIENTS AND METHODS: Fibroblasts were divided into 4 groups: (1) control, (2) UVA irradiation of 25 J/cm2, (3) UVA irradiation of 25 J/cm2 + MMPs inhibitor, and (4) 25 J/cm2 UVA irradiation + Cat G inhibitor. All treatments were repeated daily for 21 days. Western blot and ELISA was employed to detect Protein levels for Cat G, MMPs, and several smads. RESULTS: Compared to UVA-irradiated cells, the addition of MMPs inhibitor downregulated the expression of smad2, smad3, and smad4 as well as TGF-ß. The addition of Cat G inhibitor downregulated the expression of smad2, smad3, and smad4 as well as TGF-ß. These data suggest that TGF-ß/Smad signaling was decreased by inhibition of MMPs and Cat G decreased in chronically human fibroblasts which are photo-damaged. CONCLUSIONS: These results may help expand our knowledge of mechanisms mediating photoaging and is possibly instrumental to the exploration of novel anti-photoaging treatments.

Dual inhibition of cathepsin G and chymase reduces myocyte death and improves cardiac remodeling after myocardial ischemia reperfusion injury.

Early reperfusion of ischemic cardiac tissue increases inflammatory cell infiltration which contributes to cardiomyocyte death and loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Neutrophil- and mast cell-derived proteases, cathepsin G (Cat.G) and chymase, are released early after IR, but their function is complicated by potentially redundant actions and targets. This study investigated whether a dual inhibition of Cat.G and chymase influences cardiomyocyte injury and wound healing after experimental IR in mice. Treatment with a dual Cat.G and chymase inhibitor (DCCI) immediately after reperfusion blocked cardiac Cat.G and chymase activity induced after IR, which resulted in decreased immune response in the infarcted heart. Mice treated with DCCI had less myocardial collagen deposition and showed preserved ventricular function at 1 and 7 days post-IR compared with vehicle-treated mice. DCCI treatment also significantly attenuated focal adhesion (FA) complex disruption and myocyte degeneration after IR. Treatment of isolated cardiomyocytes with Cat.G or chymase significantly promoted FA signaling downregulation, myofibril degeneration and myocyte apoptosis. Conversely, treatment of cardiac fibroblasts with Cat.G or chymase induced FA signaling activation and increased their migration and differentiation to myofibroblasts. These opposite responses in cardiomyocytes and fibroblasts were blocked by treatment with DCCI. These findings show that Cat.G and chymase are key mediators of myocyte apoptosis and fibroblast migration and differentiation that play a role in adverse cardiac remodeling and function post-IR. Thus, dual targeting of neutrophil- and mast cell-derived proteases could be used as a novel therapeutic strategy to reduce post-IR inflammation and improve cardiac remodeling.

EBV Infection Empowers Human B Cells for Autoimmunity: Role of Autophagy and Relevance to Multiple Sclerosis.

The efficacy of B cell depletion therapy in multiple sclerosis indicates their central pathogenic role in disease pathogenesis. The B lymphotropic EBV is a major risk factor in multiple sclerosis, via as yet unclear mechanisms. We reported in a nonhuman primate experimental autoimmune encephalomyelitis model that an EBV-related lymphocryptovirus enables B cells to protect a proteolysis-sensitive immunodominant myelin oligodendrocyte glycoprotein (MOG) epitope (residues 40-48) against destructive processing. This facilitates its cross-presentation to autoaggressive cytotoxic MHC-E-restricted CD8+CD56+ T cells. The present study extends these observations to intact human B cells and identifies a key role of autophagy. EBV infection upregulated APC-related markers on B cells and activated the cross-presentation machinery. Although human MOG protein was degraded less in EBV-infected than in uninfected B cells, induction of cathepsin G activity by EBV led to total degradation of the immunodominant peptides MOG35-55 and MOG1-20 Inhibition of cathepsin G or citrullination of the arginine residue within an LC3-interacting region motif of immunodominant MOG peptides abrogated their degradation. Internalized MOG colocalized with autophagosomes, which can protect from destructive processing. In conclusion, EBV infection switches MOG processing in B cells from destructive to productive and facilitates cross-presentation of disease-relevant epitopes to CD8+ T cells.

Design of Potent and Selective Cathepsin G Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold.

Neutrophils are directly responsible for destroying invading pathogens via reactive oxygen species, antimicrobial peptides, and neutrophil serine proteases (NSPs). Imbalance between NSP activity and endogenous protease inhibitors is associated with chronic inflammatory disorders, and engineered inhibitors of NSPs are a potential therapeutic pathway. In this study we characterized the extended substrate specificity (P4-P1) of the NSP cathepsin G using a peptide substrate library. Substituting preferred cathepsin G substrate sequences into sunflower trypsin inhibitor-1 (SFTI-1) produced a potent cathepsin G inhibitor (Ki = 0.89 nM). Cathepsin G's P2' preference was determined by screening against a P2' diverse SFTI-based library, and the most preferred residue at P2' was combined in SFTI-1 with a preferred substrate sequence (P4-P2) and a nonproteinogenic P1 residue (4-guanidyl-l-phenylalanine) to produce a potent (Ki = 1.6 nM) and the most selective (≥360-fold) engineered cathepsin G inhibitor reported to date. This compound is a promising lead for further development of cathepsin G inhibitors targeting chronic inflammatory disorders.

Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment.

BACKGROUND: Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment. METHODS: Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe-/-and CatG-deficient mice (Apoe-/-Ctsg-/-) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy. RESULTS: Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice. CONCLUSIONS: Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects.

N-Arylacyl O-sulfonated aminoglycosides as novel inhibitors of human neutrophil elastase, cathepsin G and proteinase 3.

The balance between neutrophil serine proteases (NSPs) and protease inhibitors (PIs) in the lung is a critical determinant for a number of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis and acute lung injury. During activation at inflammatory sites, excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, PIs counteract these effects by inactivating NSPs; however, in chronic inflammatory lung diseases, there are insufficient amounts of PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP activity for the clinical management of chronic inflammatory lung diseases. In the study reported here, a panel of N-arylacyl O-sulfonated aminoglycosides was screened to identify inhibitors of the NSPs. Dose-dependent inhibitors for each individual serine protease were identified. Select compounds were found to inhibit multiple NSPs, including one lead structure that is shown to inhibit all three NSPs. Two lead compounds identified during the screen for each individual NSP were further characterized as partial mixed inhibitors of CatG. Concentration-dependent inhibition of protease-mediated detachment of lung epithelial cells is demonstrated.

ANISERP: a new serpin from the parasite Anisakis simplex.

BACKGROUND: Serine proteinase inhibitors (serpins) finely regulate serine proteinase activity via a suicide substrate-like inhibitory mechanism. In parasitic nematodes, some serpins interact with host physiological processes; however, little is known about these essential molecules in Anisakis. This article reports the gene sequencing, cloning, expression and preliminary biochemical and bioinformatically-based structural characterization of a new Anisakis serpin (ANISERP). METHODS: The full AniSerp gene was cloned by specific RACE-PCR after screening an Anisakis simplex (L3) cDNA library. For biochemical assays, the AniSerp gene was subcloned into both prokaryotic and eukaryotic vectors, and the recombinant proteins were purified. The inhibitory properties of the proteins were tested in classical biochemical assays using human serine peptidases and AMC substrates. Immunolocalization of ANISERP, theoretical structural analysis and bioinformatically-based structural modelling of the ANISERP protein were also conducted. RESULTS: The AniSerp gene was found to have 1194 nucleotides, coding for a protein of 397 amino acid residues plus a putative N-terminal signal peptide. It showed significant similarity to other nematode, arthropod and mammalian serpins. The recombinant ANISERP expressed in the prokaryotic and eukaryotic systems inhibited the human serine proteases thrombin, trypsin and cathepsin G in a concentration-dependent manner. No inhibitory activity against Factor Xa, Factor XIa, Factor XIIa, elastase, plasmin or chymotrypsin was observed. ANISERP also acted on the cysteine protease cathepsin L. ANISERP was mainly localized in the nematode pseudocoelomic fluid, somatic muscle cell bodies and intestinal cells. The findings of molecular dynamics studies suggest that ANISERP inhibits thrombin via a suicide substrate-like inhibitory mechanism, similar to the mechanism of action of mammalian coagulation inhibitors. In contrast to findings concerning human antithrombin III, heparin had no effect on ANISERP anticoagulant inhibitory activity. CONCLUSIONS: Our findings suggest that ANISERP is an internal Anisakis regulatory serpin and that the inhibitory activity against thrombin depends on a suicide substrate-like inhibitory mechanism, similar to that described for human antithrombin (AT)-III. The fact that heparin does not modulate the anticoagulant activity of ANISERP might be explained by the absence in the latter of five of the six positively charged residues usually seen at the AT-III-heparin binding site.

Miropin, a novel bacterial serpin from the periodontopathogen Tannerella forsythia, inhibits a broad range of proteases by using different peptide bonds within the reactive center loop.

All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10(4) (cathepsin G) to 7.1 × 10(5) m(-1)s(-1) (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1' site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.

Determination of cathepsin G in endometrial tissue using a surface plasmon resonance imaging biosensor with tailored phosphonic inhibitor.

OBJECTIVE: Cathepsin G is a serine peptidase whose physiological role is mainly associated with an early immune response, anti-microbial activity as well as platelet activation or hydrolysis of coagulation factors. In addition, since the activity of cathepsin G has been associated with the development of various pathological disorders, the measurement of its activity in patient samples is of high interest. Unfortunately, the usefulness of common immunological methods is limited, since they cannot distinguish between catalytically active and inactive protease. STUDY DESIGN: Here we present the application of recently developed Surface Plasmon Resonance-based biosensor for the detection of active cathepsin G in human endometrium samples. The key element of the system is based on the irreversible binding of cathepsin G to its specific phosphonic-type inhibitor immobilized on the surface of the gold chip. The concentration of cathepsin G was measured in tissue samples from the group of patients with endometriosis as well as in the control group. RESULTS: The level of cathepsin G ascertained in endometrium tissue samples was over twice as high for the group of patients suffering from endometriosis as compared to the control group, with the median values of 0.5 pmol/mg and 0.2 pmol/mg, respectively. CONCLUSION: The SPR sensor armed with a specific irreversible phosphonic inhibitor represents a highly useful tool for the determination of catalytically active cathepsin G concentration in endometrial tissue.

Inhibitors of cathepsin G: a patent review (2005 to present).

INTRODUCTION: Cathepsin G (CatG) is a neutral proteinase originating from human neutrophils. It displays a unique dual specificity (trypsin- and chymotrypsin-like); thus, its enzymatic activity is difficult to control. CatG is involved in the pathophysiology of several serious human diseases, such as chronic obstructive pulmonary disease (COPD), Crohn's disease, rheumatoid arthritis, cystic fibrosis and other conditions clinically manifested by excessive inflammatory reactions. For mentioned reasons, CatG was considered as good molecular target for the development of novel drugs. However, none of them have yet entered the market as novel therapeutic agents. AREAS COVERED: This article presents an in-depth and detailed analysis of the therapeutic potential of CatG inhibitors based on a review of patent applications and academic publishing disclosed in patents and patent applications (1991 - 2012), with several exceptions for inhibitors retrieved from academic articles. EXPERT OPINION: Among the discussed inhibitors of CatG, examples corresponding to derivatives of ß-ketophosphonic acids, aminoalkylphosphonic esters and boswellic acids (BAs) could be regarded as the most promising. The most promising one seems to be analogues of compounds of Nature's origin (peptidic and BA derivates). Nevertheless, nothing is currently known about the clinical disposition of any of the CatG inhibitors discovered so far. This latter point suggests that there is still a lot of work to do in the design of stable, pharmacologically active compounds able to specifically regulate the in vivo activity of cathepsin G.

Ovalbumin-related protein X is a heparin-binding ov-serpin exhibiting antimicrobial activities.

Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys(367)-His(368) is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.

SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G.

Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1(-/-)) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia of sB1(-/-) mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1(-/-) mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1(-/-) PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent l-leucyl-l-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy.

Effects of milk casein derived tripeptides on endothelial enzymes in vitro; a study with synthetic tripeptides.

In the fermentation of milk by certain lactic acid bacteria, casein is degraded into bioactive tripeptides shown to lower blood pressure in experimental animal models and in mildly hypertensive humans. This effect is suggested to result mainly in inhibition of angiotensin converting enzyme 1 (ACE-1).Due to the complexity of renin-angiotensin system (RAS), several other enzymes than ACE-1 can participate in the production of vasoactive components. Therefore, in the present study we investigated effects of tripeptides isoleucine-proline-proline (IPP), valine-proline-proline (VPP) and leucine-proline-proline (LPP) on some endothelial enzymes that are important in RAS or otherwise have a role in the endothelial function. The enzymes investigated were renin, chymase, neutral endopeptidase (NEP), prolyl oligopeptidase (POP), cathepsin G, endothelin converting enzyme 1 (ECE-1), and cyclooxygenase 1 and 2 (COX -1 and COX-2).The tripeptides inhibited prolyl oligopeptidase (POP) dose-dependently. IPP was the most potent inhibitor (IC50 486±95 µM). Contrary, cathepsin G was activated by IPP, VPP and LPP as well as the amino acids proline and isoleucine. The other investigated enzymes were not affected. Inhibition of POP and activation of cathepsin G do not explain the blood pressure lowering effects of the tripeptides. Thus the inhibition of ACE-1 remains the most plausible mechanism of the antihypertensive effects of the tripeptides.