DOCSLIB.ORG

Mechanism, Function, and Inhibition of Peptide Deformylase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mechanism, Function, and Inhibition of Peptide Deformylase MECHANISM, FUNCTION, AND INHIBITION OF PEPTIDE DEFORMYLASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kiet T. Nguyen ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Dehua Pei, Adviser Professor Ross Dalbey Dehua Pei Professor Sean Taylor The Ohio State Biochemistry Graduate Program ABSTRACT Peptide deformylase (PDF) was originally thought as unique and essential only to the prokaryotes and apparently was absent from the eukaryotes. In this work, two peptide deformylase homologs from the eukaryotic Plasmodium falciparum (PfPDF) and Homo Sapiens (HsPDF) were cloned and characterized. Both proteins were found to be active and contained similar properties to the Escherichia coli peptide deformylase. Expression of the PfPDF protein was detected inside the Plasmodium cell, and potent PDF inhibitors were found to reduce cell growth. The HsPDF was fused with green fluorescence protein (GFP) and revealed mitochondrial localization. Human cell growth study was carried out and was unaffected by potent PDF inhibitors. This work provides evidence that PDF indeed exists in the eukaryotes. However, the role for deformylation in humans was apparently unnecessary, and this work may validate the support for a PDF drug target, as well as providing some preliminary evidence that PDF inhibitors are not toxic to human cells. The next project involved the identification of a novel class of macrocyclic PDF inhibitors, which tailored from the traditional PDF inhibitors in being acyclic and peptide-like in nature. This new class of PDF inhibitors exhibited potent inhibition and antibacterial properties, against both the Gram-negative and Gram-positive bacteria. The cyclization effect improved the PDF inhibition selectivity and biostability. Here, this ii work may provide a lead toward more effective PDF inhibitors more resistant to proteolysis and demonstrating improved specificity. The final part of this work involved the purification and identification enzymes degrading chemotactic f-Met-Leu-Phe peptide (fMLF) released from bacteria. This area of study encompasses the PDF drug effect presented by the commensal bacteria within the mammalian intestines. N-acylpeptide hydrolase and N-acylase 1A from rat small intestine mucosal layer were isolated and identified to degrade the fMLF chemopeptide and other N-formylmethionine peptides. The results may provide support for better understanding the host defense mechanism against the commensal bacteria and to identify the potential problems that may encounter as PDF makes an attractive novel therapeutic drug target. iii Dedicated to my parents and foster parents iv ACKNOWLEDGMENTS I wish to thank my adviser, Professor Dehua Pei, for his intellectual support, professional guidance, and for having patience with me over the years. I also thank him for the environment in which he had created for a suitable professional training. I am deeply thankful for my former and present lab members for their stimulating discussion and providing support on multiple levels in the lab, especially Dr. Kirk Beebe, Mike Sweeney, Grace Zhu, Junguk Park, and Dr. Xubo Hu. As friends and colleagues, they have motivated me and provided a special place to conduct my research, an environment that I embrace with a desire to work long hours and to be able to show up the next day with the same passion. Dr. Xubo Hu was exceptionally helpful in providing the majority of the PDF inhibitors. I am grateful and dedicated my life to my wife, Valerie, who during my years of study had believed in me and provided emotional support that could only stem from a loved one. Her understanding and sacrifice had tremendously driven me to succeed and provided a place for refuge whenever I need to resolve research frustrations. This research was supported by a grant from the National Institute of Health. v VITA 1997……………………………………...B.A. Biology, Wittenberg University 1997-1999……………………………….Consultant, Materials Directorate, Wright- Patterson Air Force Research Lab 2000-present……………………………. Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Nguyen, K.T., and Pei, D. Purification and Characterization of Enzymes Involved in the Degradation of Chemotactic N-Formyl Peptides. (2005) Biochemistry, submitted. 2. Lucas, R.W., Ingasson, B.P., Krumm, B.E., Hu, X., Nguyen, K.T., Verlinde, C.L.M.J., Pei, D., and Hol, W.G.J. Rational Drug Design of Polypeptide Deformylase Inhibitors in Hemophilus Influenzae: Species Variations in Drug Design. (2005) Protein Science, in preparation. 3. Ingasson, B.P., Lucas, RW., Krumm, B.E., Hu, X., Nguyen, K.T., Verlinde, C.L.M.J., Pei, D., and Hol, W.G.J. Structural Basis for the Inhibtion of Plasmodium Falciparum Deformylase by Macrocyclic peptidomimetics. (2005) Protein Science, in preparation. 4. Nguyen, K.T., Hu, X., and Pei, D. (2004) Slow-Binding Inhibition of Peptide Deformylase by Cyclic Peptidomimetics as Revealed by a New Spectrophotometric Assay. Bioorg. Chem. 32,178-191. 5. Robien, M.A, Nguyen, K.T, Kumar, A., Hirsh, I., Turley, S., Pei, D., and Hol, W. G. J. (2004) An Improved Crystal Form of Plasmodium falciparum Peptide Deformylase. Protein Science 13, 1155-1163. 6. Hu, X., Nguyen, K.T., Vernon, C.J., Lofland, D., Moser, H.E., and Pei, D. (2004) Macrocyclic Inhibitors for Peptide Deformylase: A Structure-Activity Relationship Study of the Ring Size. J. Med. Chem. 47, 4941-4949. vi 7. Nguyen, K.T., Hu, X., Colton, C., Chakrabarti, R., Zhu, M.X., and Pei, D. (2003) Characterization of a Human Peptide Deformylase: Implications for Antibacterial Drug Design. Biochemistry 42, 9952-9958. 8. Hu, X., Nguyen, K. T., Verlinde, C. L. M. J., Hol, W. G. J., and Pei, D. (2003) Structure-Based Design of a Macrocyclic Inhibitor for Peptide Deformylase. J. Med. Chem. 46, 3771-3774. 9. Deng, H., Callender, R., Zhu, J., Nguyen, K. T., and Pei, D. (2003) Determination of the Ionization State and Catalytic Function of Glu-133 in Peptide Deformylase by Difference FTIR Spectroscopy. Biochemistry 41, 10563-10569. 10. Kumar, A., Nguyen, K. T., Srivathsan, S., Ornstein, B., Turley, S., Hirsh, I., Pei, D., and Hol, W. G. J. (2002) Crystals of Peptide Deformylase from Plasmodium falciparum Reveal Critical Characteristics of the Active Site for Drug Design. Structure 10, 357-367. 11. Bracchi-Ricard, V., Nguyen, K. T., Zhou, Y., Rajagopalan, P. T. R., Chakrabarti, D., and Pei, D. (2001) Characterization of an Eukaryotic Peptide Deformylase from Plasmodium falciparum. Arch. Biochem. Biophys. 396, 162–170. 12. Su, W., Cooper, T. M., Nguyen, K. T., Brandt, M. C., Brandelik, D. M., and McLean, D. G. (1998) Structure-optical Property Relationships of Porphyrins. Proc. SPIE, 3472, 136-143. FIELDS OF STUDY Major Field: Ohio State Biochemistry Program vii TABLE OF CONTENTS P a g e Abstract................................................................................................................................ii Dedication...........................................................................................................................iv Acknowledgments....................................................................................................... ........v Vita......................................................................................................... ............................vi List of Tables.....................................................................................................................xii List of Figures...................................................................................................................xiii List of Abbreviations.................................................................. .......................................xv Chapters: 1. General Introduction................................................................................................1 1.1 Biological Function of Peptide deformylase............ ...................................1 1.2 Peptide Deformylase in Eukaryotes.............................................................3 1.3 Structure and Mechanism...................................................................... ......5 1.4 Peptide Deformylase as Novel Drug Target................................................8 2. PDF Assays............................................................................................................15 2.1 Introduction................................................................................................15 2.2 FDH-coupled Spectrophotometry Assay (Method A)............... ...............16 2.3 AAP-coupled Spectrophotometry Assay (Method B)......................... ......17 2.4 Direct-spectrophotometry Assay (Method C)........................................ ...18 2.5 Development of a New Spectrophotometric Assay (Method D)...............19 2.5.1 Analytical Methods........................................................................19 2.5.2 Continuous PDF Assay with DPPI................................................20 2.5.3 PDF Inhibition Assay.....................................................................21 2.6 Results and Discussion......................................................................... .....21 2.6.1 Assay Design............................................................ .....................21 2.6.2 Continuous DPPI Coupled Assay..................................................22 2.6.3 Application
Load more

Recommended publications
  • Variations Through the Day of Hepatic and Muscular Cathepsin A
    Downloaded from Br. J. Nutr. (1980), 4,61 61 https://www.cambridge.org/core Variations through the day of hepatic and muscular cathepsin A (carboxypeptidase A; EC 3.4.12.2), C (dipeptidyl peptidase; EC 3.4.14.1) and D (endopeptidase D ;EC 3.4.23.5) activities and free amino acids of blood in rats: influence of feeding schedule . IP address: BY CHRISTIANE OBLED, M. ARNAL AND C. VALIN* 170.106.40.139 Laboratoire &Etude du MPtabolisme Azott!, INRA Theix-63I 10 Beaumont, France (Received 17 April 1978 - Accepted 4 January 1980) , on I. Growing rats were fed either ad lib. or with six (equal) meals offered every 4 h (from 10.00 hours). Rats 02 Oct 2021 at 01:32:10 of each group were killed at intervals of 4 h beginning at I 1.00 hours. Activities of cathepsin A (carboxy- peptidase A; EC3.4.1z.z), C (dipeptidyl peptidase; EC3.4.14.1) and D (endopeptidaseD EC3.4.23.5) were measured in liver and muscle homogenates and free amino acids in blood were determined. 2. In the rats fed ad lib. activities of carboxypeptidase A and endopeptidase D in liver and muscle showed significant variation, with maximum activity in the light period. In general, meal-feeding only caused minor differences in cathepsin activities; although significant differences occurred for carboxypeptidase A. For the latter enzyme a peak in activity occurred in the dark as well as in the light period. 3. Irrespective of the feeding schedule, the lower concentration of free essential amino acids of blood , subject to the Cambridge Core terms of use, available at occurred generally during the night period.
    [Show full text]
  • Discovery of Food-Derived Dipeptidyl Peptidase IV Inhibitory Peptides: a Review
    International Journal of Molecular Sciences Review Discovery of Food-Derived Dipeptidyl Peptidase IV Inhibitory Peptides: A Review Rui Liu 1,2,3,4 , Jianming Cheng 1,2,3,* and Hao Wu 1,2,3,* 1 Jiangsu Key Laboratory of Research and Development in Marine Bio-resource Pharmaceutics, Nanjing University of Chinese Medicine, Nanjing 210023, China; [email protected] 2 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing 210023, China 3 School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China 4 School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA * Correspondence: [email protected] (J.C.); [email protected] (H.W.); Tel.: +86-25-85811524 (J.C.); +86-25-85811206 (H.W.) Received: 21 December 2018; Accepted: 19 January 2019; Published: 22 January 2019 Abstract: Diabetes is a chronic metabolic disorder which leads to high blood sugar levels over a prolonged period. Type 2 diabetes mellitus (T2DM) is the most common form of diabetes and results from the body’s ineffective use of insulin. Over ten dipeptidyl peptidase IV (DPP-IV) inhibitory drugs have been developed and marketed around the world in the past decade. However, owing to the reported adverse effects of the synthetic DPP-IV inhibitors, attempts have been made to find DPP-IV inhibitors from natural sources. Food-derived components, such as protein hydrolysates (peptides), have been suggested as potential DPP-IV inhibitors which can help manage blood glucose levels. This review focuses on the methods of discovery of food-derived DPP-IV inhibitory peptides, including fractionation and purification approaches, in silico analysis methods, in vivo studies, and the bioavailability of these food-derived peptides.
    [Show full text]
  • 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.
    [Show full text]
  • Soybean Bioactive Peptides and Their Functional Properties
    nutrients Review Soybean Bioactive Peptides and Their Functional Properties Cynthia Chatterjee 1,2, Stephen Gleddie 2 and Chao-Wu Xiao 1,3,* 1 Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Drive, Ottawa, ON K1A 0K9, Canada; [email protected] 2 Ottawa Research & Development Centre, Central Experimental Farm, Agriculture and Agri-Food Canada, 960 Carling Avenue Building#21, Ottawa, ON K1A 0C6, Canada; [email protected] 3 Food and Nutrition Science Program, Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada * Correspondence: [email protected]; Tel.: +1-613-558-7865; Fax: +1-613-948-8470 Received: 26 July 2018; Accepted: 29 August 2018; Published: 1 September 2018 Abstract: Soy consumption has been associated with many potential health benefits in reducing chronic diseases such as obesity, cardiovascular disease, insulin-resistance/type II diabetes, certain type of cancers, and immune disorders. These physiological functions have been attributed to soy proteins either as intact soy protein or more commonly as functional or bioactive peptides derived from soybean processing. These findings have led to the approval of a health claim in the USA regarding the ability of soy proteins in reducing the risk for coronary heart disease and the acceptance of a health claim in Canada that soy protein can help lower cholesterol levels. Using different approaches, many soy bioactive peptides that have a variety of physiological functions such as hypolipidemic, anti-hypertensive, and anti-cancer properties, and anti-inflammatory, antioxidant, and immunomodulatory effects have been identified.
    [Show full text]
  • CD13/Aminopeptidase N Is a Potential Therapeutic Target for Inflammatory Disorders Chenyang Lu,*,† Mohammad A
    CD13/Aminopeptidase N Is a Potential Therapeutic Target for Inflammatory Disorders Chenyang Lu,*,† Mohammad A. Amin,* and David A. Fox* CD13/aminopeptidase N is a widely expressed ectoen- and processing of inflammatory mediators, which are im- zyme with multiple functions. As an enzyme, CD13 portant features of immune responses. In this study, we regulates activities of numerous cytokines by cleaving review evidence concerning the involvement of CD13, includ- their N-terminals and is involved in Ag processing by ing a soluble form of the molecule in inflammation and its trimming the peptides bound to MHC class II. Inde- potential as a therapeutic target in inflammatory disorders. pendent of its enzymatic activity, cell membrane CD13 functions by cross-linking–induced signal transduc- Mechanisms of CD13 functions tion, regulation of receptor recycling, enhancement of CD13 was named aminopeptidase N because of its prefer- FcgR-mediated phagocytosis, and acting as a receptor ence for binding neutral amino acids and because it removes for cytokines. Moreover, soluble CD13 has multiple N-terminal amino acids from unsubstituted oligopeptides, proinflammatory roles mediated by binding to G-pro- with the exception of peptides with proline in the penulti- tein–coupled receptors. CD13 not only modulates de- mate position (11). By cleaving N termini, CD13 regulates velopment and activities of immune-related cells, but activity of numerous hormones,cytokines,andchemokines also regulates functions of inflammatory mediators. that participate in inflammation. Moreover, CD13 is coex- Therefore, CD13 is important in the pathogenesis of pressed by MHC class II (MHC II)–bearing APCs (12), and is various inflammatory disorders. Inhibitors of CD13 involved in the trimming of MHC II–associated peptides on the have shown impressive anti-inflammatory effects, but surface of APCs (5, 13, 14) (Fig.
    [Show full text]
  • Methionine Aminopeptidase Emerging Role in Angiogenesis
    Chapter 2 Methionine Aminopeptidase Emerging role in angiogenesis Joseph A. Vetro1, Benjamin Dummitt2, and Yie-Hwa Chang2 1Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Ave., Lawrence, KS 66047, USA. 2Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, 1402 S. Grand Blvd., St. Louis, MO 63104, USA. Abstract: Angiogenesis, the formation of new blood vessels from existing vasculature, is a key factor in a number of vascular-related pathologies such as the metastasis and growth of solid tumors. Thus, the inhibition of angiogenesis has great potential as a therapeutic modality in the treatment of cancer and other vascular-related diseases. Recent evidence suggests that the inhibition of mammalian methionine aminopeptidase type 2 (MetAP2) catalytic activity in vascular endothelial cells plays an essential role in the pharmacological activity of the most potent small molecule angiogenesis inhibitors discovered to date, the fumagillin class. Methionine aminopeptidase (MetAP, EC 3.4.11.18) catalyzes the non-processive, co-translational hydrolysis of initiator N-terminal methionine when the second residue of the nascent polypeptide is small and uncharged. Initiator Met removal is a ubiquitous and essential modification. Indirect evidence suggests that removal of initiator Met by MetAP is important for the normal function of many proteins involved in DNA repair, signal transduction, cell transformation, secretory vesicle trafficking, and viral capsid assembly and infection. Currently, much effort is focused on understanding the essential nature of methionine aminopeptidase activity and elucidating the role of methionine aminopeptidase type 2 catalytic activity in angiogenesis. In this chapter, we give an overview of the MetAP proteins, outline the importance of initiator Met hydrolysis, and discuss the possible mechanism(s) through which MetAP2 inhibition by the fumagillin class of angiogenesis inhibitors leads to cytostatic growth arrest in vascular endothelial cells.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier Et Al
    US 200601 10747A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0110747 A1 Ramseier et al. (43) Pub. Date: May 25, 2006 (54) PROCESS FOR IMPROVED PROTEIN (60) Provisional application No. 60/591489, filed on Jul. EXPRESSION BY STRAIN ENGINEERING 26, 2004. (75) Inventors: Thomas M. Ramseier, Poway, CA Publication Classification (US); Hongfan Jin, San Diego, CA (51) Int. Cl. (US); Charles H. Squires, Poway, CA CI2O I/68 (2006.01) (US) GOIN 33/53 (2006.01) CI2N 15/74 (2006.01) Correspondence Address: (52) U.S. Cl. ................................ 435/6: 435/7.1; 435/471 KING & SPALDING LLP 118O PEACHTREE STREET (57) ABSTRACT ATLANTA, GA 30309 (US) This invention is a process for improving the production levels of recombinant proteins or peptides or improving the (73) Assignee: Dow Global Technologies Inc., Midland, level of active recombinant proteins or peptides expressed in MI (US) host cells. The invention is a process of comparing two genetic profiles of a cell that expresses a recombinant (21) Appl. No.: 11/189,375 protein and modifying the cell to change the expression of a gene product that is upregulated in response to the recom (22) Filed: Jul. 26, 2005 binant protein expression. The process can improve protein production or can improve protein quality, for example, by Related U.S. Application Data increasing solubility of a recombinant protein. Patent Application Publication May 25, 2006 Sheet 1 of 15 US 2006/0110747 A1 Figure 1 09 010909070£020\,0 10°0 Patent Application Publication May 25, 2006 Sheet 2 of 15 US 2006/0110747 A1 Figure 2 Ester sers Custer || || || || || HH-I-H 1 H4 s a cisiers TT closers | | | | | | Ya S T RXFO 1961.
    [Show full text]
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
    [Show full text]
  • Post Translational Modification Significance
    Post Translational Modification Significance numismatically.Depositional Rustie Vaughan never theologizes desulphurating tarnal. so denominationally or cross-index any whaups dumpishly. Brandon spiralling Senescence is related to the widespread purge of SUMO protein. The conjoint triad feature is sequence information for proteins. Although here we used sublethal antibiotics to isolate these phenotypic variants, colony size variation has been observed in mycobacteria in a variety of conditions. Moreover, three other modifications have been demonstrated to be required after fusion for modulation of RT activity. Ann Acad Med Singapore. Eukaryotic nuclei has no longer residency time points out the building allosteric effectors to toxic can occur on prevention and control by comparing prepubertal to cell motility. Epigenetic Determinants of Cancer. Protein modifications in parallel workstations and significant roles of significance of the scope of silos used for the polypeptide chain decides about posttranslational modifications mediate signaling? Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow. Reversible PTMs are particularly relevant signaling events because to offer the thrill the possibility to dynamically modulate protein activity in text response to environmental cues or internal stimuli. To borrow significant progress has been death in identifying PTMs on cardiac. The Garcia Lab has developed arguably the best precision mass spectrometry based platform for detecting and quantifying the combinatorial histone PTM code. Several PTMs, most noticeably phosphorylation, are known to directly affect metabolic enzyme activity and this aspect will be the main focus of this review. These software programs facilitate peak detection and matching, data alignment, normalization and statistical analysis. Drugs targeting an being of RNA biology we recruit to as fair-transcriptional control.
    [Show full text]
  • Formaldehyde Treatment of Proteins Enhances Proteolytic Degradation by the Endo-Lysosomal Protease Cathepsin S
    www.nature.com/scientificreports OPEN Formaldehyde treatment of proteins enhances proteolytic degradation by the endo‑lysosomal protease cathepsin S Thomas J. M. Michiels1,2, Hugo D. Meiring2, Wim Jiskoot1, Gideon F. A. Kersten1,2 & Bernard Metz2* Enzymatic degradation of protein antigens by endo‑lysosomal proteases in antigen‑presenting cells is crucial for achieving cellular immunity. Structural changes caused by vaccine production process steps, such as formaldehyde inactivation, could afect the sensitivity of the antigen to lysosomal proteases. The aim of this study was to assess the efect of the formaldehyde detoxifcation process on the enzymatic proteolysis of antigens by studying model proteins. Bovine serum albumin, β-lactoglobulin A and cytochrome c were treated with various concentrations of isotopically labelled formaldehyde and glycine, and subjected to proteolytic digestion by cathepsin S, an important endo-lysosomal endoprotease. Degradation products were analysed by mass spectrometry and size exclusion chromatography. The most abundant modifcation sites were identifed by their characteristic MS doublets. Unexpectedly, all studied proteins showed faster proteolytic degradation upon treatment with higher formaldehyde concentrations. This efect was observed both in the absence and presence of glycine, an often-used excipient during inactivation to prevent intermolecular crosslinking. Overall, subjecting proteins to formaldehyde or formaldehyde/glycine treatment results in changes in proteolysis rates, leading to an enhanced degradation speed. This accelerated degradation could have consequences for the immunogenicity and the efcacy of vaccine products containing formaldehyde- inactivated antigens. Enzymatic degradation of antigens is a crucial step in the process of acquiring cellular immunity, e.g., through the induction of antigen specifc T-helper cells or cytotoxic T-cells.
    [Show full text]
  • Formylation of Electron-Rich Aromatic Rings Mediated by Dichloromethyl Methyl Ether and Ticl4: Scope and Limitations
    Molecules 2015, 20, 5409-5422; doi:10.3390/molecules20045409 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Formylation of Electron-Rich Aromatic Rings Mediated by Dichloromethyl Methyl Ether and TiCl4: Scope and Limitations Iván Ramos-Tomillero 1,2, Marta Paradís-Bas 1,2, Ibério de Pinho Ribeiro Moreira 3,4, Josep María Bofill 2,4, Ernesto Nicolás 2,5,* and Fernando Albericio 1,2,6,7,8,* 1 Institute for Research in Biomedicine (IRB Barcelona), Barcelona 08028, Spain; E-Mails: [email protected] (I.R.-T.); [email protected] (M.P.-B.) 2 Deparment of Organic Chemistry, University of Barcelona, Barcelona 08028, Spain; E-Mail: [email protected] 3 Department of Physical Chemistry, University of Barcelona, Barcelona 08028, Spain; E-Mail: [email protected] 4 Institut de Química Teòrica i Computacional (IQTCUB), University of Barcelona, Barcelona 08028, Spain 5 Institut de Biomedicina (IBUB), University of Barcelona, Barcelona 08028, Spain 6 CIBER-BBN, Barcelona 08028, Spain 7 School of Chemistry, University of KwaZulu-Natal, Durban 4000, South Africa 8 School of Chemistry, Yachay Tech, Yachay City of Knowledge, Urcuqui 100119, Ecuador * Authors to whom correspondence should be addressed; E-Mails: [email protected] (E.N.); [email protected] or [email protected] (F.A.); Tel.: +34-93-403-7088 (F.A.). Academic Editor: Jean Jacques Vanden Eynde Received: 27 January 2015 / Accepted: 12 March 2015 / Published: 26 March 2015 Abstract: Here the aromatic formylation mediated by TiCl4 and dichloromethyl methyl ether previously described by our group has been explored for a wide range of aromatic rings, including phenols, methoxy- and methylbenzenes, as an excellent way to produce aromatic aldehydes.
    [Show full text]
  • Cloning of the Prolyl-Dipeptidyl-Peptidase from Aspergillus Oryzae
    Patentamt Europaisches ||| || 1 1| || || || || || || || || ||| || (19) J European Patent Office Office europeen des brevets (11) EP 0 897 012 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) int. CI.6: C12N 15/57, C12N 9/48, 17.02.1999 Bulletin 1999/07 C12N 1/15, C12N 1/19, (21) Application number: 97111377.4 C12P 21/06, A23J 3/30 (22) Date of filing: 05.07.1997 (84) Designated Contracting States: • Doumas, Agnes AT BE CH DE DK ES Fl FR GB GR IE IT LI LU MC 1124 Gollion (CH) NL PT SE • Affolter, Michael Designated Extension States: 1009 Pully (CH) AL LT LV RO SI • Van Den Broek Epalinges (CH) (71) Applicant: SOCIETE DES PRODUITS NESTLE S.A. (74) Representative: 1800 Vevey (CH) Straus, Alexander, Dr. Dipl.-Chem. et al KIRSCHNER & KURIG (72) Inventors: Patentanwalte • Monod, Michel Sollner Strasse 38 1 01 2 Lausanne (CH) 81 479 Munchen (DE) (54) Cloning of the prolyl-dipeptidyl-peptidase from aspergillus oryzae (57) The invention has for object the new recom- lysate obtainable by fermentation with at least a micro- binant prolyl-dipeptidyl-peptidase enzyme (DPP IV) oganism providing a prolyl-dipeptidyl-peptidase activity from Aspergillus oryzae comprising the amino-acid higher than 50 mU per ml when grown in a minimal sequence from amino acid 1 to amino acid 755 of SEQ medium containing 1 % (w/v) of wheat gluten. ID NO:2 or functional derivatives thereof, and providing a high level of hydrolysing specificity towards proteins and peptides starting with X-Pro- thus liberating dipep- tides of X-Pro type, wherein X is any amino-acid.
    [Show full text]