Loss of Chymotrypsin-Like Protease
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Natural Single-Nucleotide Deletion in Chymotrypsinogen C Gene Increases Severity of Secretagogue-Induced Pancreatitis in C57BL/6 Mice
Natural single-nucleotide deletion in chymotrypsinogen C gene increases severity of secretagogue-induced pancreatitis in C57BL/6 mice Andrea Geisz, … , Eszter Hegyi, Miklós Sahin-Tóth JCI Insight. 2019;4(14):e129717. https://doi.org/10.1172/jci.insight.129717. Research Article Gastroenterology Inflammation Genetic susceptibility to chronic pancreatitis in humans is frequently associated with mutations that increase activation of the digestive protease trypsin. Intrapancreatic trypsin activation is an early event in experimental acute pancreatitis in rodents, suggesting that trypsin is a key driver of pathology. In contrast with trypsin, the pancreatic protease chymotrypsin serves a protective function by mitigating trypsin activation through degradation. In humans, loss-of-function mutations in chymotrypsin C (CTRC) are common risk factors for chronic pancreatitis; however, the pathogenic effect of CTRC deficiency has not been corroborated in animal models yet. Here we report that C57BL/6 mice that are widely used for genetic manipulations do not express functional CTRC because of a single-nucleotide deletion in exon 2 of the Ctrc gene. We restored a functional Ctrc locus in C57BL/6N mice and demonstrated that in the Ctrc+ strain, the severity of cerulein- induced experimental acute and chronic pancreatitis was significantly ameliorated. Improved disease parameters were associated with reduced intrapancreatic trypsin activation, suggesting a causal link between CTRC-mediated trypsinogen degradation and protection against pancreatitis. -
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. -
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. -
Diapause in the Mosquito Culex Pipiens Evokes a Metabolic Switch from Blood Feeding to Sugar Gluttony
Diapause in the mosquito Culex pipiens evokes a metabolic switch from blood feeding to sugar gluttony Rebecca M. Robich* and David L. Denlinger† Department of Entomology, Ohio State University, 318 West 12th Avenue, Columbus, OH 43210 Contributed by David L. Denlinger, September 12, 2005 A key characteristic of overwintering dormancy (diapause) in the Although the physiological and ecological aspects of blood and mosquito Culex pipiens is the switch in females from blood feeding sugar feeding in diapausing C. pipiens have been well described, the to sugar gluttony. We present evidence demonstrating that genes molecular events that contribute to this metabolic decision have not encoding enzymes needed to digest a blood meal (trypsin and a been explored. In this study, we used suppressive subtractive chymotrypsin-like protease) are down-regulated in diapause-des- hybridization (SSH) to isolate three clones linked to this metabolic tined females, and that concurrently, a gene associated with the decision: fatty acid synthase, trypsin, and chymotrypsin-like serine accumulation of lipid reserves (fatty acid synthase) is highly up- protease. We then used these clones to probe the metabolic path- regulated. As the females then enter diapause, fatty acid synthase ways associated with the mosquito’s metabolic decision to enter and is only sporadically expressed, and expression of trypsin and terminate diapause. Because both short day length and low tem- chymotrypsin-like remains undetectable. Late in diapause (2–3 perature program diapause in C. pipiens, we also distinguished months at 18°C), the genes encoding the digestive enzymes begin between temperature and photoperiodic effects. We concluded to be expressed as the female prepares to take a blood meal upon that the short-day programming of diapause results in the down- the termination of diapause. -
Wsn 40 (2016) 147-162 Eissn 2392-2192
Available online at www.worldscientificnews.com WSN 40 (2016) 147-162 EISSN 2392-2192 Utilization of mulberry leaves treated with seed powder cowpea, Vigna unguiculata (L) for feeding the fifth instar larvae of silkworm, Bombyx mori (L) (Race: PM x CSR2) Vitthalrao B. Khyade1,*, Atharv Atul Gosavi2 1Department of Zoology, Shardabai Pawar Mahila Mahavidyalaya, Shardanagar Tal. Baramati; Dist. Pune - 413115, India 2Agriculture Development Trust Agri Polytechnic, Sharadanagar, Malegaon Colony, Tal: Baramati, Dist: Pune. PIN: 413115 Maharashtra, India *E-mail address: [email protected] ABSTRACT The present attempt was to screen the changes in the cocoon parameters; silk filament parameters and activities of biochemical reactions catalyzed by the midgut enzymes fifth instsr larvae of silkworm fed with mulberry leaves treated with aqueous solution of seed powder of Cowpeas (Vigna unguiculata). The cowpea seed powder was dissolved in distilled water and diluted to 2.5%, 5%, 7.5%, and 10% concentrations. Fresh mulberry leaves were dipped in each concentration of aqueous solution of cowpea seed powder for half an hour. 1000 ml solution was used for 100 grams of mulberry leaves. Treated mulberry leaves were drained off completely and then used for feeding. The mulberry leaves were fed five times per day at the rate of 100 grams per 100 larvae for each time. Untreated group of larvae were feed with untreated mulberry leaves. Water treated group of larvae were feed with water treated mulberry leaves. The experimental groups of larvae were feed with feed separately with 2.5 percent cowpea treated; 5.00 percent cowpea treated; 7.5 percent cowpea treated and 10.00 percent cowpea treated mulberry leaves. -
Monospecific Inhibitors
Supplemental Material can be found at: http://www.jbc.org/content/suppl/2012/04/16/M112.354332.DC1.html THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 24, pp. 20290–20300, June 8, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Monospecific Inhibitors Show That Both Mannan-binding Lectin-associated Serine Protease-1 (MASP-1) and -2 Are Essential for Lectin Pathway Activation and Reveal Structural Plasticity of MASP-2*□S Received for publication, February 17, 2012, and in revised form, March 30, 2012 Published, JBC Papers in Press, April 16, 2012, DOI 10.1074/jbc.M112.354332 Dávid Héja‡1,2, Veronika Harmat§¶1, Krisztián Fodor**, Matthias Wilmanns**, József Dobóʈ3, Katalin A. Kékesi‡‡§§, Péter Závodszkyʈ, Péter Gálʈ4,5, and Gábor Pál‡3,4,6 From the ‡Department of Biochemistry, Eötvös Loránd University, 1/C Pázmány Péter Street, H-1117, Budapest, Hungary, §Institute of Chemistry, Eötvös Loránd University, 1/A Pázmány Péter Street, H-1117, Budapest, Hungary, ¶Protein Modeling Research Group, Hungarian Academy of Sciences, Eötvös Loránd University, 1/A Pázmány Péter Street, H-1117, Budapest, Hungary, **European Molecular Downloaded from Biology Laboratory-Hamburg, c/o DESY, Building 25a, Notkestrasse 85, 22603 Hamburg, Germany, ʈInstitute ofEnzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 29. Karolina street, H-1113, Budapest, Hungary, ‡‡Department of Physiology and Neurobiology of Eötvös Loránd University, 1/C Pázmány Péter Street, H-1117, Budapest, Hungary, and §§Proteomics Group of the Biology Institute of Eötvös Loránd University, 1/C Pázmány Péter Street, H-1117, Budapest, Hungary Background: MASP-1 is considered as auxiliary, whereas MASP-2 is considered as a key protease in lectin-pathway activation. -
Digestive Enzymes: the Key to Optimum Health
Education Article Digestive Enzymes: The Key to Optimum Health Digestion is essential for good health. Unlocking nutrients from foods is a complex process. In order to break down food we rely on optimal levels and function of a special set of proteins called digestive enzymes. These proteins are found in the saliva and also in the small intestines. Without the combined actions of our digestive enzymes we would simply be unable to absorb many nutrients that we need to maintain good health. This is why reduced levels of digestive enzymes can be linked to a wide-range of symptoms within the gut and beyond. Digestive enzymes, along with stomach acid, play a crucial role in the initial stages of digestion, i.e. the breaking down of the food that we eat. The main classes of human digestive enzymes include proteases, lipases and carbohydrases, which respectively break down the macronutrients protein, fats and carbohydrates. If we do not efficiently digest these foods then vital nutrients such as essential fats, vitamins, minerals and phytonutrients cannot be absorbed. What is more, undigested or partially digested food passes through into the large intestines and is fermented by the resident colonic bacteria, causing unpleasant symptoms such as bloating and flatulence and contributing to a toxic bowel. Naturopaths believe toxicity within the bowel is the root of all disease. We do not make digestive enzymes for every type of food that we eat, such as gluten and phytic acid found in some grains and cereals and lactose, a sugar found in milk. This means our bodies may find it difficult to digest these types of foods. -
Partial Characterization of Hepatopancreatic and Extracellular
Aquaculture International Archimer June 2011, Volume 19, Number 3, Pages 445-457 http://archimer.ifremer.fr http://dx.doi.org/10.1007/s10499-010-9360-5 © Springer Science+Business Media B.V. 2010 The original publication is available at http://www.springerlink.com is available on the publisher Web site Web publisher the on available is Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated Octopus maya R. Martínez1, R. Sántos2, A. Álvarez3, G. Cuzón4, L. Arena5, M. Mascaró5, C. Pascual5 and C. Rosas5, * 1 División de Posgrado, FMVZ, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico 2 authenticated version authenticated Departamento de Nutrición, FMVZ, Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico - 3 Unidad de Ciencias Biológicas, UJAT, Villahermosa, Tabasco, Mexico 4 Ifremer, Tahiti, French Polynesia 5 Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, Universidad Nacional Autónoma de México, Puerto de abrigo s/n, Sisal, Yucatan, Mexico *: Corresponding author : C. Rosas, email address : [email protected] Abstract: Abstract Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9–10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction on enzyme activity of 70 and 20% was observed in HP and GJ extracts, respectively when protease inhibitor Pepstatin A was used. -
Characterization and Functionalization of Suckerin-12 Protein
CHARACTERIZATION AND FUNCTIONALIZATION OF SUCKERIN-12 PROTEIN HYDROGELS Dissertation Submitted to The School of Engineering of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Doctor of Philosophy in Engineering By Chelsea Buck, M.S. UNIVERSITY OF DAYTON Dayton, Ohio December 2018 CHARACTERIZATION AND FUNCTIONALIZATION OF SUCKERIN-12 PROTEIN HYDROGELS Name: Buck, Chelsea Carolyn APPROVED BY: Kristen K. Comfort, Ph. D. Donald A. Klosterman, Ph. D. Advisory Committee Chairman Committee Member Associate Professor Associate Professor Chemical and Materials Engineering Chemical and Materials Engineering Margaret F. Pinnell, Ph. D. Patrick B. Dennis, Ph. D. Committee Member Committee Member Associate Dean Research Scientist Mechanical and Aerospace Engineering Air Force Research Laboratory Robert J. Wilkens, Ph.D., P.E. Eddy M. Rojas, Ph.D., M.A., P.E. Associate Dean for Research and Innovation Dean Professor School of Engineering School of Engineering ii © Copyright by Chelsea Carolyn Buck All rights reserved 2018 iii ABSTRACT CHARACTERIZATION AND FUNCTIONALIZATION OF SUCKERIN-12 PROTEIN HYDROGELS Name: Buck, Chelsea Carolyn University of Dayton Advisor: Dr. Kristen K. Comfort Previous research of suckerin proteins identified in the sucker ring teeth of cephalopods have impressive mechanical properties and behave as thermoplastic materials. In this research, one isoform of suckerin protein, suckerin-12 was explored as a mechanically robust material. The protein was isolated and recombinantly expressed in E. coli. Gram-scale quantities of pure protein were expressed and purified to create enzymatically crosslinked hydrogels. Exposure to select salt anion conditions caused the hydrogels to contract significantly, at rates highly dependent upon the anion present in the buffer, which followed a trend modeled by the Hofmeister Series of anions. -
Trypsinogen Isoforms in the Ferret Pancreas Eszter Hegyi & Miklós Sahin-Tóth
www.nature.com/scientificreports OPEN Trypsinogen isoforms in the ferret pancreas Eszter Hegyi & Miklós Sahin-Tóth The domestic ferret (Mustela putorius furo) recently emerged as a novel model for human pancreatic Received: 29 June 2018 diseases. To investigate whether the ferret would be appropriate to study hereditary pancreatitis Accepted: 25 September 2018 associated with increased trypsinogen autoactivation, we purifed and cloned the trypsinogen isoforms Published: xx xx xxxx from the ferret pancreas and studied their functional properties. We found two highly expressed isoforms, anionic and cationic trypsinogen. When compared to human cationic trypsinogen (PRSS1), ferret anionic trypsinogen autoactivated only in the presence of high calcium concentrations but not in millimolar calcium, which prevails in the secretory pathway. Ferret cationic trypsinogen was completely defective in autoactivation under all conditions tested. However, both isoforms were readily activated by enteropeptidase and cathepsin B. We conclude that ferret trypsinogens do not autoactivate as their human paralogs and cannot be used to model the efects of trypsinogen mutations associated with human hereditary pancreatitis. Intra-pancreatic trypsinogen activation by cathepsin B can occur in ferrets, which might trigger pancreatitis even in the absence of trypsinogen autoactivation. Te digestive protease precursor trypsinogen is synthesized and secreted by the pancreas to the duodenum where it becomes activated to trypsin1. Te activation process involves limited proteolysis of the trypsinogen activation peptide by enteropeptidase, a brush-border serine protease specialized for this sole purpose. Te activation peptide is typically an eight amino-acid long N-terminal sequence, which contains a characteristic tetra-aspartate motif preceding the activation site peptide bond, which corresponds to Lys23-Ile24 in human trypsinogens. -
Fibrinolysis Influences SARS-Cov-2 Infection in Ciliated Cells
bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425801; this version posted January 8, 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. 1 Fibrinolysis influences SARS-CoV-2 infection in ciliated cells 2 3 Yapeng Hou1, Yan Ding1, Hongguang Nie1, *, Hong-Long Ji2 4 5 1Department of Stem Cells and Regenerative Medicine, College of Basic Medical Science, China Medical 6 University, Shenyang, Liaoning 110122, China. 2Department of Cellular and Molecular Biology, University 7 of Texas Health Science Center at Tyler, Tyler, TX 75708, USA. 8 9 *Address correspondence to [email protected] 10 11 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425801; this version posted January 8, 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. 12 Abstract 13 Rapid spread of COVID-19 has caused an unprecedented pandemic worldwide, and an inserted furin site 14 in SARS-CoV-2 spike protein (S) may account for increased transmissibility. Plasmin, and other host 15 proteases, may cleave the furin site of SARS-CoV-2 S protein and subunits of epithelial sodium channels ( 16 ENaC), resulting in an increment in virus infectivity and channel activity. As for the importance of ENaC in 17 the regulation of airway surface and alveolar fluid homeostasis, whether SARS-CoV-2 will share and 18 strengthen the cleavage network with ENaC proteins at the single-cell level is urgently worthy of consideration. -
Specificity of a Polyclonal Fecal Elastase ELISA for CELA3
RESEARCH ARTICLE Specificity of a Polyclonal Fecal Elastase ELISA for CELA3 Frank Ulrich Weiss, Christoph Budde, Markus M. Lerch* University Medicine Greifswald, Department of Medicine A, Ferdinand Sauerbruch-Str., D-17475 Greifswald, Germany * [email protected] a11111 Abstract Introduction Elastase is a proteolytic pancreatic enzyme that passes through the gastrointestinal tract undergoing only limited degradation. ELISA tests to determine stool elastase concentra- tions have therefore been developed for the diagnosis of exocrine pancreatic insufficiency. OPEN ACCESS Five different isoforms of pancreatic elastase (CELA1, CELA2A, CELA2B, CELA3A, Citation: Weiss FU, Budde C, Lerch MM (2016) CELA3B) are encoded in the human genome. We have investigated three different poly- Specificity of a Polyclonal Fecal Elastase ELISA for CELA3. PLoS ONE 11(7): e0159363. doi:10.1371/ clonal antisera that are used in a commercial fecal elastase ELISA to determine their speci- journal.pone.0159363 ficity for different pancreatic elastase isoforms. Editor: Keping Xie, The University of Texas MD Anderson Cancer Center, UNITED STATES Material and Methods Received: October 9, 2015 Different polyclonal rabbit antisera against human elastase peptides (BIOSERV Diagnos- Accepted: July 2, 2016 tics GmbH, Germany) were tested by Western blot analysis of human pancreatic juice, in HEK-293 cells expressing Elastase constructs, and in the protein content of porcine pancre- Published: July 26, 2016 atin, used for treatment of exocrine pancreatic insufficiency. Copyright: © 2016 Weiss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits Results unrestricted use, distribution, and reproduction in any In human pancreatic juice the polyclonal antisera detected proteins at the corresponding medium, provided the original author and source are size of human pancreatic elastase isoforms (~29kDa).