New Approaches for Dissecting Protease Functions to Improve Probe Development and Drug Discovery
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Solutions to Detect Apoptosis and Pyroptosis
Powerful Tools for In vitro Fluorescent Imaging in Cancer Research Jackie Carville May 11, 2016 About Us Products and Services • Custom Assay Services – Immunoassay design and development – Antibody purification/conjugation • Consumable Reagents – ELISA Solutions – Cell-permeant Fluorescent Probes for detection of cell death • Products for research use only. Not for use in diagnostic procedures. ELISA Solutions • ELISA Wash Buffer • PBS • Substrates • Stop Solutions • Plates • Coating Buffers • Blocking Buffers • Conjugate Stabilizers • Assay Diluents • Sample Diluents Custom Services • Antibody Conjugation • Immunoassay Design • Immunoassay Development • Lyophilization • Plate Coating Overview of ICT’s Fluorescent Probes FOR DETECTION OF: • Active caspases • Caspases/Cathepsins in real time • Mitochondrial Health • Cytotoxicity • Serine Proteases Today’s Agenda • FLICA • FLISP • Magic Red • Cell Viability • Mito PT • Oxidative Stress • Cytotoxicity FLICA • FLICA – Fluorescent Labeled Inhibitor of Caspases • In vitro, whole cell detection of caspase activity in apoptotic or caspase-positive cells • Available in green, red, and far red assays FLICA Cancer Research Applications • FLICA is the most accurate and sensitive method of apoptosis detection • Can identify four stages of cell death in one sample • Detect poly caspase activity or individual caspase enzymes How FLICA A. INACTIVE CASPASE (ZYMOGEN) works… prodomain B. CASPASE PROCESSING FMK Fluorescent Reporter YVAD tag C. ACTIVATED CASPASE (HETERO-TETRAMER) D. BINDING OF FLICA Sample -
The Osteoclast-Associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1
CANCER RESEARCH57. 5386-5390. December I, 19971 The Osteoclast-associated Protease Cathepsin K Is Expressed in Human Breast Carcinoma1 Amanda J. Littlewood-Evans,' Graeme Bilbe, Wayne B. Bowler, David Farley, Brenda Wlodarski, Toshio Kokubo, Tetsuya Inaoka, John Sloane, Dean B. Evans, and James A. Gallagher Novartis Pharina AG, CH-4002 Base!. Switzerland (A. I. L-E., G. B., D. F., D. B. El; Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology 1W. B. B., B. W., J. A. G.J, and Department of Pathology If. S.), University of Liverpool, Liverpool 5.69 3BX, United Kingdom [W. B. B., B. W., J. S., J. A. G.]; and Takarazuka Research Institute, Novartis Pharma lid., Takarazuka 665, Japan IT. K., T. LI ABSTRACT (ZR75—l,Hs578Tduct, BT474, ZR75—30,BT549, and T-47D), adenocarci noma (MDA-MB-231, SK-BR3, MDA-MB-468, and BT2O), and breast car Human cathepsin K is a novel cysteine protease previously reported cinoma[MVLNandMTLN,transfectedMCF-7-derivedclonesobtainedfrom to be restricted in its expression to osteoclasts. Immunolocalization of Dr. J. C. Nicolas (Unit de Recherches sur Ia Biochemie des Steroides Insern cathepsin K in breast tumor bone metastases revealed that the invading U58, Montpelier, France), and MCF-7]. The MCF-7 cells were obtained from breast cancer cells expressed this protease, albeit at a lower intensity two different sources, ATCC and Dr. Roland Schuele (Tumor Klinik, Freiburg, than in osteoclasts. In situ hybridization and immunolocalization stud Germany). These are indicated in the figure legend as clone 1 (ATCC) and ies were subsequently conducted to demonstrate cathepsin K mRNA clone 2 (Dr. -
Design of a Highly Selective Quenched Activity-Based Probe
Article pubs.acs.org/JACS Design of a Highly Selective Quenched Activity-Based Probe and Its Application in Dual Color Imaging Studies of Cathepsin S Activity Localization † † † † ∥ Kristina Oresic Bender, Leslie Ofori, Wouter A. van der Linden, Elliot D. Mock, Gopal K. Datta, ∥ § † † ∥ # Somenath Chowdhury, Hao Li, Ehud Segal, Mateo Sanchez Lopez, Jonathan A. Ellman, , ⊥ † ‡ § † ⊥ Carl G. Figdor, Matthew Bogyo,*, , , and Martijn Verdoes*, , † ‡ § Departments of Pathology, Microbiology and Immunology, and Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305, United States ∥ Department of Chemistry, University of California-Berkeley, Berkeley, California 94720, United States ⊥ Department of Tumor Immunology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, 6500 HB Nijmegen, The Netherlands *S Supporting Information ABSTRACT: The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. -
Ubiquitin-Mediated Proteolysis the Nobel Prize in Chemistry for 2004 Is
Advanced information on the Nobel Prize in Chemistry, 6 October 2004 Information Department, P.O. Box 50005, SE-104 05 Stockholm, Sweden Phone: +46 8 673 95 00, Fax: +46 8 15 56 70, E-mail: [email protected], Website: www.kva.se Ubiquitin-mediated proteolysis The Nobel Prize in Chemistry for 2004 is shared between three scientists who have made fundamental discoveries concerning how cells regulate the breakdown of intracellular proteins with extreme specificity as to target, time and space. Aaron Ciechanover, Avram Hershko and Irwin Rose together discovered ubiquitin- mediated proteolysis, a process where an enzyme system tags unwanted proteins with many molecules of the 76-amino acid residue protein ubiquitin. The tagged proteins are then transported to the proteasome, a large multisubunit protease complex, where they are degraded. Numerous cellular processes regulated by ubiquitin-mediated proteolysis include the cell cycle, DNA repair and transcription, protein quality control and the immune response. Defects in this proteolysis have a causal role in many human diseases, including a variety of cancers. Fig. 1 Ubiquitin-mediated proteolysis and its many biological functions 2 Introduction Eukaryotic cells, from yeast to human, contain some 6000 to 30000 protein-encoding genes and at least as many proteins. While much attention and research had been devoted to how proteins are synthesized, the reverse process, i.e. how proteins are degraded, long received little attention. A pioneer in this field was Schoenheimer, who in 1942 published results from isotope tracer techniques indicating that proteins in animals are continuously synthesized and degraded and therefore are in a dynamic state (Schoenheimer, 1942). -
Towards Therapy for Batten Disease
Towards therapy for Batten disease Mariana Catanho da Silva Vieira MRC Laboratory for Molecular Cell Biology University College London PhD Supervisor: Dr Sara E Mole A thesis submitted for the degree of Doctor of Philosophy University College London September 2014 Declaration I, Mariana Catanho da Silva Vieira, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract The gene underlying the classic neurodegenerative lysosomal storage disorder (LSD) juvenile neuronal ceroid lipofuscinosis (JNCL) in humans, CLN3, encodes a polytopic membrane spanning protein of unknown function. Several studies using simpler models have been performed in order to further understand this protein and its pathological mechanism. Schizosaccharomyces pombe provides an ideal model organism for the study of CLN3 function, due to its simplicity, genetic tractability and the presence of a single orthologue of CLN3 (Btn1p), which exhibits a functional profile comparable to its human counterpart. In this study, this model was used to explore the effect of different mutations in btn1 as well as phenotypes arising from complete deletion of the gene. Different btn1 mutations have different effects on the protein function, underlining different phenotypes and affecting the levels of expression of Btn1p. So far, there is no cure for JNCL and therefore it is of great importance to identify novel lead compounds that can be developed for disease therapy. To identify these compounds, a drug screen with btn1Δ cells based on their sensitivity to cyclosporine A, was developed. Positive hits from the screen were validated and tested for their ability to rescue other specific phenotypes also associated with the loss of btn1. -
House Dust Mites: Ecology, Biology, Prevalence, Epidemiology and Elimination Muhammad Sarwar
Chapter House Dust Mites: Ecology, Biology, Prevalence, Epidemiology and Elimination Muhammad Sarwar Abstract House dust mites burrow cheerfully into our clothing, pillowcases, carpets, mats and furniture, and feed on human dead skin cells by breaking them into small particles for ingestion. Dust mites are most common in asthma allergens, and some people have a simple dust allergy, but others have an additional condition called atopic dermatitis, often stated to as eczema by reacting to mites with hideous itching and redness. The most common type of dust mites are Dermatophagoides farinae Hughes (American house dust mite) and Dermatophagoides pteronyssinus Trouessart (European house dust mite) of family Pyroglyphidae (Acari), which have been associated with dermatological and respiratory allergies in humans such as eczema and asthma. A typical house dust mite measures 0.2–0.3 mm and the body of mite has a striated cuticle. A mated female house dust mite can live up to 70 days and lays 60–100 eggs in the last 5 weeks of life, and an average life cycle is 65–100 days. In a 10-week life span, dust mite produces about 2000 fecal particles and an even larger number of partially digested enzyme-covered dust particles. They feed on skin flakes from animals, including humans and on some mold. Notably, mite’s gut contains potent digestive enzymes peptidase 1 that persist in their feces and are major induc- ers of allergic reactions, but its exoskeleton can also contribute this. Allergy testing by a physician can determine respiratory or dermatological symptoms to undergo allergen immunotherapy, by exposing to dust mite extracts for “training” immune system not to overreact. -
Secreted Metalloproteinase ADAMTS-3 Inactivates Reelin
The Journal of Neuroscience, March 22, 2017 • 37(12):3181–3191 • 3181 Cellular/Molecular Secreted Metalloproteinase ADAMTS-3 Inactivates Reelin Himari Ogino,1* Arisa Hisanaga,1* XTakao Kohno,1 Yuta Kondo,1 Kyoko Okumura,1 Takana Kamei,1 Tempei Sato,2 Hiroshi Asahara,2 Hitomi Tsuiji,1 Masaki Fukata,3 and Mitsuharu Hattori1 1Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, Japan, 2Department of Systems BioMedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8510, Japan, and 3Division of Membrane Physiology, Department of Molecular and Cellular Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer’s disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo. Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. -
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. -
What Is a Protease?
What is a Protease? Proteases (or peptidases) are enzymes secreted by animals for a number of physiological processes, among which is the Dr. Rolando A. Valientes digestion of feed protein. Regional Category Manager- Animals normally secrete suffi - Eubiotics / RONOZYME ProAct cient amount of enzymes to ade - Asia Pacific, DSM quately digest enough of their [email protected] feed so that they grow and remain healthy under normal conditions, such as those found in the wild. Any increased needs for protein (amino acids) for more rapid growth due to improved genetics has been traditionally met by adding more protein (or synthetic amino acids) into the feed. This was facilitated by a relative low cost for most protein-rich ingredi - Dr. Katrine Pontoppidan ents, such as soybean meal, and Research Scientist synthetic amino acids, such as Novozymes L-lysine HCL. Thus, an exogenous [email protected] protease (as a feed supplement) was not considered essential; that is, until recently. size, the rate of passage of feed Today we face not only the prob - through the digestive tract, the lem of feeding animals of continu - age of the animal, and its physio - ously increasing genetic potential logical/health condition. All of (this requires diets increasingly these variables are rather difficult richer in amino acids), but also an to control, but supplementing unprecedented rise in ingredient animal feeds with extra enzymes prices, leaving very small (if any) is rather easy if it can be done in a margin for profitability. Thus, it profitable way. has been deemed essential to seek ways to improve the nutritive Up until recently, any protease value of existing ingredients activity in commercial enzyme reducing feed cost. -
Crystal Structure of Cathepsin X: a Flip–Flop of the Ring of His23
st8308.qxd 03/22/2000 11:36 Page 305 Research Article 305 Crystal structure of cathepsin X: a flip–flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease Gregor Guncar1, Ivica Klemencic1, Boris Turk1, Vito Turk1, Adriana Karaoglanovic-Carmona2, Luiz Juliano2 and Dušan Turk1* Background: Cathepsin X is a widespread, abundantly expressed papain-like Addresses: 1Department of Biochemistry and v mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia and 2Departamento de well as carboxy-dipeptidase activity and shares a similar activity profile with Biofisica, Escola Paulista de Medicina, Rua Tres de cathepsin B. The latter has been implicated in normal physiological events as Maio 100, 04044-020 Sao Paulo, Brazil. well as in various pathological states such as rheumatoid arthritis, Alzheimer’s disease and cancer progression. Thus the question is raised as to which of the *Corresponding author. E-mail: [email protected] two enzyme activities has actually been monitored. Key words: Alzheimer’s disease, carboxypeptidase, Results: The crystal structure of human cathepsin X has been determined at cathepsin B, cathepsin X, papain-like cysteine 2.67 Å resolution. The structure shares the common features of a papain-like protease enzyme fold, but with a unique active site. The most pronounced feature of the Received: 1 November 1999 cathepsin X structure is the mini-loop that includes a short three-residue Revisions requested: 8 December 1999 insertion protruding into the active site of the protease. The residue Tyr27 on Revisions received: 6 January 2000 one side of the loop forms the surface of the S1 substrate-binding site, and Accepted: 7 January 2000 His23 on the other side modulates both carboxy-monopeptidase as well as Published: 29 February 2000 carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. -
Cells T+ HLA-DR + Processing in Human CD4 Cathepsin S
Cathepsin S Regulates Class II MHC Processing in Human CD4 + HLA-DR+ T Cells This information is current as Cristina Maria Costantino, Hidde L. Ploegh and David A. of September 26, 2021. Hafler J Immunol 2009; 183:945-952; Prepublished online 24 June 2009; doi: 10.4049/jimmunol.0900921 http://www.jimmunol.org/content/183/2/945 Downloaded from References This article cites 47 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/183/2/945.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Cathepsin S Regulates Class II MHC Processing in Human CD4؉ HLA-DR؉ T Cells1 Cristina Maria Costantino,* Hidde L. Ploegh,† and David A. Hafler2* Although it has long been known that human CD4؉ T cells can express functional class II MHC molecules, the role of lysosomal proteases in the T cell class II MHC processing and presentation pathway is unknown. -
The Involvement of Cysteine Proteases and Protease Inhibitor Genes in the Regulation of Programmed Cell Death in Plants
The Plant Cell, Vol. 11, 431–443, March 1999, www.plantcell.org © 1999 American Society of Plant Physiologists The Involvement of Cysteine Proteases and Protease Inhibitor Genes in the Regulation of Programmed Cell Death in Plants Mazal Solomon,a,1 Beatrice Belenghi,a,1 Massimo Delledonne,b Ester Menachem,a and Alex Levine a,2 a Department of Plant Sciences, Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904, Israel b Istituto di Genetica, Università Cattolica S.C., Piacenza, Italy Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and bio- chemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress in- duced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine pro- tease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhib- itors was ineffective. A glutathione S-transferase–cystatin fusion protein was used to purify and characterize the in- duced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.