Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma
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The Role of Streptococcal and Staphylococcal Exotoxins and Proteases in Human Necrotizing Soft Tissue Infections
toxins Review The Role of Streptococcal and Staphylococcal Exotoxins and Proteases in Human Necrotizing Soft Tissue Infections Patience Shumba 1, Srikanth Mairpady Shambat 2 and Nikolai Siemens 1,* 1 Center for Functional Genomics of Microbes, Department of Molecular Genetics and Infection Biology, University of Greifswald, D-17489 Greifswald, Germany; [email protected] 2 Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, CH-8091 Zurich, Switzerland; [email protected] * Correspondence: [email protected]; Tel.: +49-3834-420-5711 Received: 20 May 2019; Accepted: 10 June 2019; Published: 11 June 2019 Abstract: Necrotizing soft tissue infections (NSTIs) are critical clinical conditions characterized by extensive necrosis of any layer of the soft tissue and systemic toxicity. Group A streptococci (GAS) and Staphylococcus aureus are two major pathogens associated with monomicrobial NSTIs. In the tissue environment, both Gram-positive bacteria secrete a variety of molecules, including pore-forming exotoxins, superantigens, and proteases with cytolytic and immunomodulatory functions. The present review summarizes the current knowledge about streptococcal and staphylococcal toxins in NSTIs with a special focus on their contribution to disease progression, tissue pathology, and immune evasion strategies. Keywords: Streptococcus pyogenes; group A streptococcus; Staphylococcus aureus; skin infections; necrotizing soft tissue infections; pore-forming toxins; superantigens; immunomodulatory proteases; immune responses Key Contribution: Group A streptococcal and Staphylococcus aureus toxins manipulate host physiological and immunological responses to promote disease severity and progression. 1. Introduction Necrotizing soft tissue infections (NSTIs) are rare and represent a more severe rapidly progressing form of soft tissue infections that account for significant morbidity and mortality [1]. -
Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis
Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis Ghada Al-Salih1, Nawwar Al-Attar2,3, Sandrine Delbosc1, Liliane Louedec1, Elisabeth Corvazier1, Ste´phane Loyau1, Jean-Baptiste Michel1,3, Dominique Pidard1,4, Xavier Duval5, Olivier Meilhac1,3,6* 1 INSERM U698, Paris, France, 2 Cardiovascular Surgery Department, Bichat Hospital, AP-HP, Paris, France, 3 University Paris Diderot, Sorbonne Paris Cite´, Paris, France, 4 Institut National des Sciences du Vivant, Centre National de la Recherche Scientifique, Paris, France, 5 INSERM CIC 007, Paris, France, 6 Bichat Stroke Centre, AP-HP, Paris, France Abstract Aims: Infective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization. Methods and Results: IE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO). -
Trypsin Inhibitor from Glycine Max (Soybean) (T6522)
Trypsin inhibitor from Glycine max (soybean) Cell Culture Tested Product Number T 6522 Storage Temperature 2-8 °C Product Description Precautions and Disclaimer CAS Number: 9035-81-8 For Laboratory Use Only. Not for drug, household or Extinction Coefficient: E1% = 9.94 (280 nm, other uses. pH 7.6 buffer) pI: 4.51 Preparation Instructions Synonyms: Kunitz Trypsin Inhibitor, Tia1, STI, and Trypsin inhibitor is soluble in water and phosphate SBT1 buffers at 10 mg/ml. It is soluble in balanced salt solutions (1 mg/ml) and in serum-free media. This product is cell culture tested and is appropriate Solutions at concentrations higher than 10 mg/ml may for use in cell culture applications. It is extensively be hazy and have a yellow to amber color. dialyzed against water. After dialysis, sodium phosphate buffer, pH 7.6, is added, and the inhibitor is Storage/Stability lyophilized. The final product consists of about 90% A 10 mg/ml sterile-filtered solution stored for greater protein and 10% sodium phosphate buffer salts (by than 3 years at 2-8 °C showed no loss in trypsin mass). inhibition activity. Solutions are stable in frozen aliquots at -20 °C, but freeze-thaw cycles should be 2 Soybean trypsin inhibitor was first isolated by Kunitz. avoided. This protein is reversibly denatured by short Several other related inhibitors are also found in heating to 80 °C and irreversibly inhibited by heating to 3 soybeans. Trypsin inhibitor from soybeans is a 90 °C.3 monomeric protein containing 181 amino acid residues in a single polypeptide chain crosslinked by two 4,5,6 Procedure disulfide bridges. -
Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases
International Journal of Molecular Sciences Review Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases Peter Goettig Structural Biology Group, Faculty of Molecular Biology, University of Salzburg, Billrothstrasse 11, 5020 Salzburg, Austria; [email protected]; Tel.: +43-662-8044-7283; Fax: +43-662-8044-7209 Academic Editor: Cheorl-Ho Kim Received: 30 July 2016; Accepted: 10 November 2016; Published: 25 November 2016 Abstract: Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications. Keywords: secreted protease; sequon; N-glycosylation; O-glycosylation; core glycan; enzyme kinetics; substrate recognition; flexible loops; Michaelis constant; turnover number 1. -
The Plasmin–Antiplasmin System: Structural and Functional Aspects
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Bern Open Repository and Information System (BORIS) Cell. Mol. Life Sci. (2011) 68:785–801 DOI 10.1007/s00018-010-0566-5 Cellular and Molecular Life Sciences REVIEW The plasmin–antiplasmin system: structural and functional aspects Johann Schaller • Simon S. Gerber Received: 13 April 2010 / Revised: 3 September 2010 / Accepted: 12 October 2010 / Published online: 7 December 2010 Ó Springer Basel AG 2010 Abstract The plasmin–antiplasmin system plays a key Plasminogen activator inhibitors Á a2-Macroglobulin Á role in blood coagulation and fibrinolysis. Plasmin and Multidomain serine proteases a2-antiplasmin are primarily responsible for a controlled and regulated dissolution of the fibrin polymers into solu- Abbreviations ble fragments. However, besides plasmin(ogen) and A2PI a2-Antiplasmin, a2-Plasmin inhibitor a2-antiplasmin the system contains a series of specific CHO Carbohydrate activators and inhibitors. The main physiological activators EGF-like Epidermal growth factor-like of plasminogen are tissue-type plasminogen activator, FN1 Fibronectin type I which is mainly involved in the dissolution of the fibrin K Kringle polymers by plasmin, and urokinase-type plasminogen LBS Lysine binding site activator, which is primarily responsible for the generation LMW Low molecular weight of plasmin activity in the intercellular space. Both activa- a2M a2-Macroglobulin tors are multidomain serine proteases. Besides the main NTP N-terminal peptide of Pgn physiological inhibitor a2-antiplasmin, the plasmin–anti- PAI-1, -2 Plasminogen activator inhibitor 1, 2 plasmin system is also regulated by the general protease Pgn Plasminogen inhibitor a2-macroglobulin, a member of the protease Plm Plasmin inhibitor I39 family. -
Development and Binding Characteristics of Phosphonate Inhibitors of Spla Protease from Staphylococcus Aureus
Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus Ewa Burchacka,1 Michal Zdzalik,2 Justyna-Stec Niemczyk,3 Katarzyna Pustelny,3 Grzegorz Popowicz,4 Benedykt Wladyka,3,5 Adam Dubin,3 Jan Potempa,2 Marcin Sienczyk,1 Grzegorz Dubin,2,5* and Jozef Oleksyszyn1 1Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wroclaw University of Technology, Wroclaw, Poland 2Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 3Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 4Max-Planck Institute for Biochemistry, NMR Group, Martinsried, Germany 5Malopolska Centre of Biotechnology, Krakow, Poland Received 24 September 2013; Revised 27 November 2013; Accepted 2 December 2013 DOI: 10.1002/pro.2403 Published online 4 December 2013 proteinscience.org Abstract: Staphylococcus aureus is responsible for a variety of human infections, including life- threatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in par- ticular SplA protease, remain poorly characterized. Here, we report development of specific inhibi- tors of SplA protease. The design, synthesis, and activity of a series of a-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding P P modes of the high-affinity compounds Cbz-Phe -(OC6H424-SO2CH3)2 and Suc-Val-Pro-Phe - (OC6H5)2 are revealed by high-resolution crystal structures of complexes with the protease. Sur- prisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of a-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases. -
Characterization of a Bowman–Birk Type Trypsin Inhibitor Purified From
www.nature.com/scientificreports OPEN Characterization of a Bowman–Birk type trypsin inhibitor purifed from seeds of Solanum surattense Abhijeet P. Herwade1, Sainath S. Kasar1,2, Niraj R. Rane3, Shadab Ahmed4, Jaswinder Singh Maras5 & Pankaj K. Pawar6* A Bowman–Birk type trypsin inhibitor protein (SSTI) from seeds of the medicinal plant Solanum surattense was isolated, purifed and characterized. SSTI showed a single band on SDS-PAGE corresponding to 11.4 kDa molecular weight. It is a glycoprotein (2.8% glycosylation) that diferentially interacted with trypsin and chymotrypsin in a concentration-dependent manner. Its peptide sequence is similar to other Bowman–Birk type protease inhibitors found in Glycine max and Phaseolus acutifolius. The inhibitory activity was stable over a wide range of pH (1–10) and temperatures (10–100° C). Far-UV Circular Dichroism (CD) studies showed that SSTI contains β sheets (~ 23%) and α helix (~ 6%) and demonstrated structural stability at wide pH and high temperature. The kinetic analysis revealed a noncompetitive (mixed) type nature of SSTI and low inhibitor constant (Ki) −8 values (16.6 × 10 M) suggested strong inhibitory activity. Isothermal titration calorimetric analysis revealed its high afnity towards trypsin with dissociation constant (Kd) 2.28 µM. Biotic stress induces the generation and accumulation of phenolic compounds and pathogenesis-related (PR) proteins which subsequently prevent an invasion of pests like insects and microbial pathogens1. Most of the plant PR proteins are acid-soluble, low molecular weight and protease enzyme inhibitors 2,3. Protease inhibitors are mainly harbored by four plant families’ viz. Fabaceae, Gramineae, Leguminosae, and Solanaceae4,5. -
Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases
International Journal of Molecular Sciences Review Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases Emma L. Carroll 1,†, Mariarca Bailo 2,†, James A. Reihill 1 , Anne Crilly 2 , John C. Lockhart 2, Gary J. Litherland 2, Fionnuala T. Lundy 3 , Lorcan P. McGarvey 3, Mark A. Hollywood 4 and S. Lorraine Martin 1,* 1 School of Pharmacy, Queen’s University, Belfast BT9 7BL, UK; [email protected] (E.L.C.); [email protected] (J.A.R.) 2 Institute for Biomedical and Environmental Health Research, School of Health and Life Sciences, University of the West of Scotland, Paisley PA1 2BE, UK; [email protected] (M.B.); [email protected] (A.C.); [email protected] (J.C.L.); [email protected] (G.J.L.) 3 Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University, Belfast BT9 7BL, UK; [email protected] (F.T.L.); [email protected] (L.P.M.) 4 Smooth Muscle Research Centre, Dundalk Institute of Technology, A91 HRK2 Dundalk, Ireland; [email protected] * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are Citation: Carroll, E.L.; Bailo, M.; characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to Reihill, J.A.; Crilly, A.; Lockhart, J.C.; Litherland, G.J.; Lundy, F.T.; persistent inflammation, infection and dysregulated protease activity. -
Association Between Production of Fibrinolysin Exudative Epidermitis in Pigs
Acta vet. scand. 1997, 38, 295-297. Brief Communication Association Between Production of Fibrinolysin and Virulence of Staphylococcus hyicus in Relation to Exudative Epidermitis in Pigs Staphylococcus hyicus is the causative agent of of these is fibrinolysin or staphylokinase. exudative epidermitis (EE) in pigs, character Staphylokinase is a protein produced by many ized by a generalized infection of the skin with S. aureus and S. hyicus strains (Devriese et al. greasy exudation and exfoliation (L'Ecuyer 1978, Devriese & Kerckhove 1980) that binds 1966). S. hyicus is a natural part ofthe skin flora to plasminogen and converts it into plasmin that of healthy pigs worldwide (Wegener 1992), and dissolves proteins and fibrinogen. The potential several different strains may simultaneously rple of fibrinolysin in pathogenesis of bacterial colonize the same pig (Wegener 1993a). Both infections is not known, but it has been specu virulent and avirulent strains can be present si lated that it might help the bacteria in obtaining multaneously on diseased piglets (Wegener et amino acids and in colonization by dissolving al. 1993), and virulent strains can be isolated fibrinogen and other proteins. Production offib from healthy carriers (Devriese 1977, Park & rinolysin has previously been described in S. Kang 1988). The pathogenesis of EE has only hyicus from pigs in different countries in Eu been studied in a limited number of studies, but rope (Devriese et al. 1978). This study de EE most likely occurs as a consequence of skin scribes the occurrence of fibrinolysin produc trauma that exposes the dermis and facilitates tion among virulent and avirulent isolates of S. -
Uterine-Associated Serine Protease Inhibitors Stimulate Deoxyribonucleic Acid Synthesis in Porcine Endometrial Glandular Epithelial Cells of Pregnancy 1
BIOLOGY OF REPRODUCTION 61, 380±387 (1999) Uterine-Associated Serine Protease Inhibitors Stimulate Deoxyribonucleic Acid Synthesis in Porcine Endometrial Glandular Epithelial Cells of Pregnancy 1 Lokenga Badinga, Frank J. Michel, and Rosalia C.M. Simmen2 Animal Molecular and Cell Biology Interdisciplinary Concentration, Department of Animal Science, University of Florida, Gainesville, Florida 32611-0910 ABSTRACT Consistent with this, uteri from mammalian species with distinct placentation types express common classes of pro- Protease inhibitors are major secretory components of the tease inhibitors (e.g., tissue inhibitors of metalloproteases, Downloaded from https://academic.oup.com/biolreprod/article/61/2/380/2734487 by guest on 24 September 2021 mammalian uterus that are thought to mediate pregnancy-as- TIMPs) as well as distinct ones (e.g., secretory leukocyte sociated events primarily by regulating the activity of proteolytic protease inhibitor, SLPI, and uterine plasmin/trypsin inhib- enzymes. In the present study, we examined the mitogenic po- tentials of two serine protease inhibitors, namely secretory leu- itor, UPTI) [4, 8, 9]. Since embryos from all species, re- kocyte protease inhibitor (SLPI) and uterine plasmin/trypsin in- gardless of placentation type, exhibit invasive properties hibitor (UPTI) in primary cultures of glandular epithelial (GE) when placed into ectopic sites [10], the limiting of blasto- cells isolated from early pregnant (Day 12) pig endometrium, cyst invasiveness, albeit to varying extents, is most likely -
Detection of Complexes Between Prostate-Specific Antigen and Protease Inhibitors in Plasma Ulf-Håkan Stenman1*
Clinical Chemistry 56:12 1895–1896 (2010) Citation Classic Detection of Complexes between Prostate-Specific Antigen and Protease Inhibitors in Plasma Ulf-Håkan Stenman1* Featured Article: Stenman UH, Leinonen J, Alfthan H, but not eliminated, by measuring PSA–ACT and total Rannikko S, Tuhkanen K, Alfthan O. A complex between PSA simultaneously with a double-label assay, by cor- ␣ prostate-specific antigen and 1-antichymotrypsin is the recting for the nonspecific background measured sep- major form of prostate-specific antigen in serum of arately in each sample, and by using a monoclonal an- patients with prostatic cancer: assay of the complex im- tibody to the PSA–ACT complex (2). proves clinical sensitivity for cancer. Cancer Res The reason for devoting so much effort to the ac- 1991;51:222–6.2 curate measurement of PSA–ACT was that it is the Prostate-specific antigen (PSA)3 had been in clin- most cancer-specific form of PSA. Other PSA com- ical use for several years when we encountered a prob- plexes may account for up to 10% of total PSA, but ␣ lem with 2 samples that did not give expected results contrary to PSA–ACT, the proportions of PSA- 1- ␣ upon dilution. To explore this finding, we subjected protease inhibitor and PSA- 2-macroglobulin are the samples to gel filtration and found that a major part higher in BPH than in cancer. Thus, measurement of of immunoreactive PSA had a molecular size of about all complexed forms of PSA together is inferior to mea- 90 kD rather than the expected size of 30 kD. -
Receptor Mediated Catabolism of Plasminogen Activators
RECEPTOR MEDIATED CATABOLISM OF PLASMINOGEN ACTIVATORS by Philip George Grimsley Thesis submitted for the degree of Doctor of Philosophy from The University of New South Wales School of Medical Sciences 2009 PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Grimsley First name: Philip Other name/s: George Abbreviation for degree as given in the University calendar: PhD School: School of Medical Sciences Faculty: Medicine Title: Receptor Mediated Catabolism of Plasminogen Activators Abstract 350 words maximum: (PLEASE TYPE) Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2-macroglobulin, apolipoproteins, β-amyloid precursor protein, and a number of serpin-enzymes complexes, including PA•PAI-1 complexes.