TMPRSS2 and Furin Are Both Essential for Proteolytic Activation and Spread of SARS-Cov-2 in Human Airway Epithelial Cells and Pr
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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. -
(12) Patent Application Publication (10) Pub. No.: US 2016/0346364 A1 BRUNS Et Al
US 2016.0346364A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0346364 A1 BRUNS et al. (43) Pub. Date: Dec. 1, 2016 (54) MEDICAMENT AND METHOD FOR (52) U.S. Cl. TREATING INNATE IMMUNE RESPONSE CPC ........... A61K 38/488 (2013.01); A61K 38/482 DISEASES (2013.01); C12Y 304/23019 (2013.01); C12Y 304/21026 (2013.01); C12Y 304/23018 (71) Applicant: DSM IPASSETS B.V., Heerlen (NL) (2013.01); A61K 9/0053 (2013.01); C12N 9/62 (2013.01); A23L 29/06 (2016.08); A2ID 8/042 (72) Inventors: Maaike Johanna BRUINS, Kaiseraugst (2013.01); A23L 5/25 (2016.08); A23V (CH); Luppo EDENS, Kaiseraugst 2002/00 (2013.01) (CH); Lenneke NAN, Kaiseraugst (CH) (57) ABSTRACT (21) Appl. No.: 15/101,630 This invention relates to a medicament or a dietary Supple (22) PCT Filed: Dec. 11, 2014 ment comprising the Aspergillus niger aspergilloglutamic peptidase that is capable of hydrolyzing plant food allergens, (86). PCT No.: PCT/EP2014/077355 and more particularly, alpha-amylase/trypsin inhibitors, thereby treating diseases due to an innate immune response S 371 (c)(1), in humans, and/or allowing to delay the onset of said (2) Date: Jun. 3, 2016 diseases. The present invention relates to the discovery that (30) Foreign Application Priority Data the Aspergillus niger aspergilloglutamic peptidase is capable of hydrolyzing alpha-amylase/trypsin inhibitors that are Dec. 11, 2013 (EP) .................................. 13196580.8 present in wheat and related cereals said inhibitors being strong inducers of innate immune response. Furthermore, Publication Classification the present invention relates to a method for hydrolyzing alpha-amylase/trypsin inhibitors comprising incubating a (51) Int. -
A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated. -
Efficacy and Safety of the MC4R Agonist Setmelanotide in POMC Deficiency Obesity: a Phase 3 Trial
Efficacy and Safety of the MC4R Agonist Setmelanotide in POMC Deficiency Obesity: A Phase 3 Trial Karine Clément,1,2 Jesús Argente,3 Allison Bahm,4 Hillori Connors,5 Kathleen De Waele,6 Sadaf Farooqi,7 Greg Gordon,5 James Swain,8 Guojun Yuan,5 Peter Kühnen9 1Sorbonne Université, INSERM, Nutrition and Obesities Research Unit, Paris, France; 2Assistance Publique Hôpitaux de Paris, Pitié- Salpêtrière Hospital, Nutrition Department, Paris, France; 3Department of Pediatrics & Pediatric Endocrinology Universidad Autónoma de Madrid University, Madrid, Spain; 4Peel Memorial Hospital, Toronto, Canada; 5Rhythm Pharmaceuticals, Inc., Boston, MA; 6Ghent University Hospital, Ghent, Belgium; 7Wellcome-MRC Institute of Metabolic Science and NIHR Cambridge Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom; 8HonorHealth Bariatric Center, Scottsdale, AZ; 9Institute for Experimental Pediatric Endocrinology Charité Universitätsmedizin Berlin, Berlin, Germany Melanocortin Signaling Is Crucial for Regulation of Body Weight1,2 • Body weight is regulated by the hypothalamic central melanocortin pathway • In response to leptin signaling, POMC is produced in POMC neurons and is cleaved by protein convertase subtilisin/kexin type 1 into α-MSH and β-MSH • α-MSH and β-MSH bind to the MC4R, which decreases food intake and increases energy expenditure, thereby promoting a reduction in body weight Hypothalamus AgRP/NPY Neuron LEPR Hunger AgRP Food Intake ADIPOSE Weight TISSUE MC4R- Energy Expressing Expenditure MC4R Neuron LEPTIN PCSK1 BLOOD-BRAIN BARRIER POMC α-MSH LEPR POMC Neuron AgRP, agouti-related protein; LEPR, leptin receptor; MC4R, melanocortin 4 receptor; MSH, melanocyte-stimulating hormone; NPY, neuropeptide Y; PCSK1, proprotein convertase subtilisin/kexin type 1; POMC, proopiomelanocortin. 2 1. Yazdi et al. -
Steroid-Dependent Regulation of the Oviduct: a Cross-Species Transcriptomal Analysis
University of Kentucky UKnowledge Theses and Dissertations--Animal and Food Sciences Animal and Food Sciences 2015 Steroid-dependent regulation of the oviduct: A cross-species transcriptomal analysis Katheryn L. Cerny University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Cerny, Katheryn L., "Steroid-dependent regulation of the oviduct: A cross-species transcriptomal analysis" (2015). Theses and Dissertations--Animal and Food Sciences. 49. https://uknowledge.uky.edu/animalsci_etds/49 This Doctoral Dissertation is brought to you for free and open access by the Animal and Food Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Animal and Food Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. -
TMPRSS2 and Furin Are Both Essential for Proteolytic Activation of SARS-Cov-2 in Human Airway Cells
Research Article TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells Dorothea Bestle1,*, Miriam Ruth Heindl1,*, Hannah Limburg1,*, Thuy Van Lam van2 , Oliver Pilgram2, Hong Moulton3, David A Stein3 , Kornelia Hardes2,4, Markus Eickmann1,5, Olga Dolnik1,5 , Cornelius Rohde1,5, Hans-Dieter Klenk1, Wolfgang Garten1, Torsten Steinmetzer2, Eva Bottcher-Friebertsh¨ auser¨ 1 The novel emerged SARS-CoV-2 has rapidly spread around the broad range of mammalian and avian species, causing respiratory world causing acute infection of the respiratory tract (COVID-19) or enteric diseases. CoVs have a major surface protein, the spike (S) that can result in severe disease and lethality. For SARS-CoV-2 to protein, which initiates infection by receptor binding and fusion of enter cells, its surface glycoprotein spike (S) must be cleaved at the viral lipid envelope with cellular membranes. Like fusion two different sites by host cell proteases, which therefore rep- proteins of many other viruses, the S protein is activated by cellular resent potential drug targets. In the present study, we show that S proteases. Activation of CoV S is a complex process that requires can be cleaved by the proprotein convertase furin at the S1/S2 proteolytic cleavage of S at two distinct sites, S1/S2 and S29 (Fig 1), site and the transmembrane serine protease 2 (TMPRSS2) at the generating the subunits S1 and S2 that remain non-covalently S29 site. We demonstrate that TMPRSS2 is essential for activation linked (1, 2, 3). The S1 subunit contains the receptor binding do- of SARS-CoV-2 S in Calu-3 human airway epithelial cells through main, whereas the S2 subunit is membrane-anchored and harbors antisense-mediated knockdown of TMPRSS2 expression. -
Serine Proteases with Altered Sensitivity to Activity-Modulating
(19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants. -
CDH12 Cadherin 12, Type 2 N-Cadherin 2 RPL5 Ribosomal
5 6 6 5 . 4 2 1 1 1 2 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 A A A A A A A A A A A A A A A A A A A A C C C C C C C C C C C C C C C C C C C C R R R R R R R R R R R R R R R R R R R R B , B B B B B B B B B B B B B B B B B B B , 9 , , , , 4 , , 3 0 , , , , , , , , 6 2 , , 5 , 0 8 6 4 , 7 5 7 0 2 8 9 1 3 3 3 1 1 7 5 0 4 1 4 0 7 1 0 2 0 6 7 8 0 2 5 7 8 0 3 8 5 4 9 0 1 0 8 8 3 5 6 7 4 7 9 5 2 1 1 8 2 2 1 7 9 6 2 1 7 1 1 0 4 5 3 5 8 9 1 0 0 4 2 5 0 8 1 4 1 6 9 0 0 6 3 6 9 1 0 9 0 3 8 1 3 5 6 3 6 0 4 2 6 1 0 1 2 1 9 9 7 9 5 7 1 5 8 9 8 8 2 1 9 9 1 1 1 9 6 9 8 9 7 8 4 5 8 8 6 4 8 1 1 2 8 6 2 7 9 8 3 5 4 3 2 1 7 9 5 3 1 3 2 1 2 9 5 1 1 1 1 1 1 5 9 5 3 2 6 3 4 1 3 1 1 4 1 4 1 7 1 3 4 3 2 7 6 4 2 7 2 1 2 1 5 1 6 3 5 6 1 3 6 4 7 1 6 5 1 1 4 1 6 1 7 6 4 7 e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m -
Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma
Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma Joakim E. Swedberg,‡† Guojie Wu,§† Tunjung Mahatmanto,‡# Thomas Durek,‡ Tom T. Caradoc-Davies,∥ James C. Whisstock,§* Ruby H.P. Law§* and David J. Craik‡* ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia §ARC Centre of Excellence in Advanced Molecular Imaging, Department of Biochemistry and Molecular Biology, Biomedical Discovery Institute, Monash University, VIC 3800, Australia. ∥Australian Synchrotron, 800 Blackburn Road, Clayton, Melbourne, VIC 3168, Australia. †J.E.S. and G.W. contributed equally to this work. Keywords: Antifibrinolytics; Fibrinolysis; Inhibitors; Peptides; Plasmin ABSTRACT Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses and non-selective inhibition mechanisms. These deficiencies warrant the development of a new generation highly potent and selective fibrinolysis inhibitors. Here we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent (Ki = 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects. 1 INTRODUCTION The physiological process of fibrinolysis regulates the dissolution of blood clots and thrombosis. -
Coagulation Factors Directly Cleave SARS-Cov-2 Spike and Enhance Viral Entry
bioRxiv preprint doi: https://doi.org/10.1101/2021.03.31.437960; this version posted April 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry. Edward R. Kastenhuber1, Javier A. Jaimes2, Jared L. Johnson1, Marisa Mercadante1, Frauke Muecksch3, Yiska Weisblum3, Yaron Bram4, Robert E. Schwartz4,5, Gary R. Whittaker2 and Lewis C. Cantley1,* Affiliations 1. Meyer Cancer Center, Department of Medicine, Weill Cornell Medical College, New York, NY, USA. 2. Department of Microbiology and Immunology, Cornell University, Ithaca, New York, USA. 3. Laboratory of Retrovirology, The Rockefeller University, New York, NY, USA. 4. Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA. 5. Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, USA. *Correspondence: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.03.31.437960; this version posted April 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Summary Coagulopathy is recognized as a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. Other host proteases, including TMPRSS2, are recognized to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing viral entry. -
Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (Enac) by Combining Current Measurements with Detection of Cleavage Fragments
Journal of Visualized Experiments www.jove.com Video Article Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments Matteus Krappitz1, Christoph Korbmacher1, Silke Haerteis1 1 Institut für Zelluläre und Molekulare Physiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) Correspondence to: Silke Haerteis at [email protected] URL: http://www.jove.com/video/51582 DOI: doi:10.3791/51582 Keywords: Biochemistry, Issue 89, two-electrode voltage-clamp, electrophysiology, biotinylation, Xenopus laevis oocytes, epithelial sodium channel, ENaC, proteases, proteolytic channel activation, ion channel, cleavage sites, cleavage fragments Date Published: 7/5/2014 Citation: Krappitz, M., Korbmacher, C., Haerteis, S. Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments. J. Vis. Exp. (89), e51582, doi:10.3791/51582 (2014). Abstract The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole- cell current (ΔIami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. -
Cell Surface–Anchored Serine Proteases in Cancer Progression and Metastasis
Cancer and Metastasis Reviews (2019) 38:357–387 https://doi.org/10.1007/s10555-019-09811-7 Cell surface–anchored serine proteases in cancer progression and metastasis Carly E. Martin1,2 & Karin List1,2 Published online: 16 September 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Over the last two decades, a novel subgroup of serine proteases, the cell surface–anchored serine proteases, has emerged as an important component of the human degradome, and several members have garnered significant attention for their roles in cancer progression and metastasis. A large body of literature describes that cell surface–anchored serine proteases are deregulated in cancer and that they contribute to both tumor formation and metastasis through diverse molecular mechanisms. The loss of precise regulation of cell surface–anchored serine protease expression and/or catalytic activity may be contributing to the etiology of several cancer types. There is therefore a strong impetus to understand the events that lead to deregulation at the gene and protein levels, how these precipitate in various stages of tumorigenesis, and whether targeting of selected proteases can lead to novel cancer intervention strategies. This review summarizes current knowledge about cell surface–anchored serine proteases and their role in cancer based on biochemical characterization, cell culture–based studies, expression studies, and in vivo experiments. Efforts to develop inhibitors to target cell surface–anchored serine proteases in cancer therapy will also be summarized. Keywords Type II transmembrane serine proteases . Cancer . Matriptase . Hepsin . TMPRSS2 . TMPRSS3 . TMPRSS4 . Prostasin . Testisin 1 Introduction PRSS31, transmembrane tryptase, and transmembrane prote- ase γ1) is expressed in cells of hematopoietic origin and has The class of serine proteases contains 175 predicted members been studied most extensively in mast cells [2].