DOCSLIB.ORG

Monitoring Proteolytic Efficiency of Engineered Trypsin Via Chymotrypsinogen Activity Assa Y

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Monitoring Proteolytic Efficiency of Engineered Trypsin Via Chymotrypsinogen Activity Assa Y MONITORING PROTEOLYTIC EFFICIENCY OF ENGINEERED TRYPSIN VIA CHYMOTRYPSINOGEN ACTIVITY ASSA Y A thesis submitted to the faculty of f t 5 San Francisco State University 3 ^ In partial fulfillment of Z^O 1 ? The requirements for C t t m The Degree , V f f Master of Science In Chemistry Concentration in Biochemistry By Rodolfo Villa San Francisco, California San Francisco State University July 2019 CERTIFICATION OF APPROVAL I certify that I have read MONITORING PROTEOLYTIC EFFICIENCY OF ENGINEERED TRYPSIN VIA CHYMOTRYPSINOGEN ACTIVITY ASSAY by Rodolfo Villa, and that in my opinion this work meets the criteria for approving a thesis submitted in partial fulfillment of the requirement for the degree Master of Science in Chemistry: Biochemistry at San Francisco State University. MONITORING PROTEOLYTIC EFFICIENCY OF ENGINEERED TRYPSIN VIA CHYMOTRYPSINOGENACTIVITYASSAY Rodolfo Villa San Francisco, California 2019 Protease therapeutics have been on the rise and have gained recognition for having diverse clinical applications. One major complication faced is the abundance of protease inhibitors that serve to regulate proteolytic activity resulting in a short therapeutic half-life. Previous research with the model serine protease trypsin showed that residues 39 and 60 play a key role in protease: inhibitor binding. Compared to wild type, four trypsin single variants (Y39A, Y39F, K60A, K60V) which were all catalytically similar using a synthetic substrate displayed altered.sensitivity towards bovine pancreatic trypsin inhibitor (BPTI) compared to wild type. In order to ascertain the viability of these engineered variants for inhibitor resistance in vivo, the interactions between naturally occurring macro molecular substrates were evaluated in this study. Variant Y39A displayed the highest kcat/KM (pM'1Min-1) at 3.34 ± 0.06, while K60V displayed at 0.58 ± 0.01. Variant Y39A activated the most chymotrypsinogen (Cg) with a total 30% activation 1.48 ± 0.05 jvM (out of 5 pM) while K60V had the lowest with 14% or 0.70 ± 0.04 pM. With 20 nM BPTI, the K60A variant had an activation drop of 49% while K60V dropped by 16%. At 30 nM, 50 nM and 100nM BPTI, the Y39F variant displayed greater activation than wild-type and the K60 variants in the presence of BPTI. At higher concentrations of BPTI, the K60 variants s have become more sensitive to inhibition, along with having lower kcat/KM and less total chymotrypsin activation. Overall K60 variants seemed to be less proteolytically efficient towards macromolecular substrates with less resistance towards inhibition, while Y39F displayed promising results in both studies. I certify that the Abstract is a correct presentation of the content of this ) Date ACKNOWLEDGEMENTS I would like to thank the support of the Student Enrichment Opportunities program that allowed me to take part in the Bridges to the Doctorate program which provided me with not only with financial assistance but allowed my development as a scientist. I extend my sincerest gratitude to my principal investigator of this work Dr. Teaster Baird, who took me into his lab and provided with mentorship support with my studes and research, for his patience, motivation, and immense knowledge that has been shared. I could not have asked for a better mentor and advisor. I would also like to thank the rest of my thesis committee: Dr. Ray Esquerra, and Dr. George Gassner, for their encouragement and support. Last, I would like to thank my parents Rodolfo Villa and Candelaria Villa for supporting me on every move and decision I have made throughout my life. TABLE OF CONTENTS List of Tables.......................................................................................................... viii List of Figures.......................................................................................................... ix List of Equations...................................................................................................... x 1. Introduction.......................................................................................................... 1 Protease Classification................................................................................ 2 Serine Proteases.......................................................................................... 4 Catalytic Mechanism....................................................................................7 Protease Inhibition....................................................................................... 8 Protease Therapeutics............................................................................... 12 Protease Engineering................................................................................. 15 2. Experimental design........................................................................................... 17 Kinetic Characterization Using Macromolecular Substrates.................... 17 Inhibition Characterization..........................................................................19 3. Methods..............................................................................................................20 Generation of Trypsin Variants...................................................................20 Expression of Wild Type Trypsin............................................................... 20 Fast Protein Liquid Chromatography (FPLC)........................................... 21 Zymogen Activation....................................................................................21 Separation of Trypsin from Trypsinogen................................................... 22 Active Site Titration.....................................................................................22 Kinetic Characterization............................................................................. 23 BPTI Inhibition Kinetics.............................................................................. 23 Data Analysis..............................................................................................24 4. Discussion..........................................................................................................25 YPDS-Zeocin plates/Bacterial growth....................................................... 25 Large Scale Expression............................................................................. 25 Fast Protein Liquid Chromatography (FPLC)........................................... 26 Zymogen Activation................................................................................... 27 Separation of Trypsin from Trypsinogen................................................... 28 Active Site Titration.................................................................... 29 Kinetic Characterization............................................................................31 BPTI Inhibition Kinetics...............................................................................35 5. Future Directions.................................................................................................43 6. Citations..............................................................................................................44 vii LIST OF TABLES Table Page 1. Protease Classifications....................................................................................... 3 2. Major Features of Serine Protease Inhibitors.....................................................9 3. FDA- Approved Protease Drugs.........................................................................14 4 Active Site Titration Wild-Type and Variants........................................................30 5. Kinetic Parameters Kcat/Km and total amount of active chymotrypsin 34 6. Ratio of active chymotrypsin in the presence of BPTI...................................... 37 LIST OF FIGURES Figures Page 1. Structure of Wild-Type Trypsin........................................................................... 5 2. Oxyanion H ole................................................... 6 3. Trypsin and BPTI Inhibitor complex.................................................................. 11 4. Z-Gly-Pro-Arg-p-nitroanilide (Z-GPR-pNA)..................................................... 17 5. Kinetic Characterization Scheme...................................................................... 18 6. SDS-PAGE Protein Activation....................................................... 28 7. PAB Column SDS-PAGE...................................................................................29 8. Fluorescence Standard Curve.......................................................................... 30 9. Kinetic Characterization Control.........................................................................32 10. Kinetic Characterization Results .............................................................. 34 11.. BPTI Inhibition Kinetics...................................................................................37 12. Wild-Type Trypsin S1 ’ Pocket, Kinetic, Inhibition Results.............................38 13. Y39A Trypsin S1 ’ Pocket,b) Kinetic, c) Inhibition Results.............................. 39 14. Y39F Trypsin S1 ’ Pocket,b) Kinetic, c) Inhibition Results...............................40 15. K60A Trypsin S1’ Pocket,b) Kinetic, c) Inhibition Results...............................41 16. K60V Trypsin S1’ Pocket,b) Kinetic, c) Inhibition Results...............................42 EQUATIONS Equations Figures 1. Exponential Burst Equation..............................................................................
Load more

Recommended publications
  • Molecular Markers of Serine Protease Evolution
    The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages.
    [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]
  • Progress Report the Interrelationships of the Pancreatic Enzymes
    Gut: first published as 10.1136/gut.13.5.398 on 1 May 1972. Downloaded from Gut, 1972, 13, 398-412 Progress report The interrelationships of the pancreatic enzymes The quantitative interrelationships between the different functional types of pancreatic enzymes has been the subject of much experimental investiga- tion, but the results are conflicting and the inferences remain controversial. Recent improvements in techniques have provided new and more reliable means of measuring enzyme activities and it therefore seems pertinent to review the current state of knowledge about the factors which determine selective production of the pancreatic enzymes. Pavlovl, quoting the studies of his pupil Walther in dogs, described rapid 'adaptive' changes in the amounts of individual pancreatic enzymes secreted in response to changes in the predominant type of food in a single meal. Later studies supported Pavlov's hypothesis that the production of individual enzymes, or groups ofenzymes, could be selectively modified, but changed the emphasis by indicating that adaptation of pancreatic enzyme secretion might require prolonged dietary modification, for periods ranging from hours to weeks2'3'4. http://gut.bmj.com/ The converse hypothesis, that pancreatic enzymes were secreted 'in parallel', was proposed during the early years of the present century by Babkin5,6 on the basis of experiments in dogs with pancreatic fistulae and was later supported by studies of pancreatic secretion in other species, including man. In order to disentangle the current state of the evidence for the two on September 29, 2021 by guest. Protected copyright. conflicting hypotheses, the interrelationships between the pancreatic enzymes within the pancreatic-secreting cells, in the secreted juice, and within the small intestinal lumen are considered separately, since different factors determine the relative increase or decrease of individual enzymes or groups of enzymes in the three compartments.
    [Show full text]
  • Studies of Structure and Function of Tripeptidyl-Peptidase II
    Till familj och vänner List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I. Eriksson, S.; Gutiérrez, O.A.; Bjerling, P.; Tomkinson, B. (2009) De- velopment, evaluation and application of tripeptidyl-peptidase II se- quence signatures. Archives of Biochemistry and Biophysics, 484(1):39-45 II. Lindås, A-C.; Eriksson, S.; Josza, E.; Tomkinson, B. (2008) Investiga- tion of a role for Glu-331 and Glu-305 in substrate binding of tripepti- dyl-peptidase II. Biochimica et Biophysica Acta, 1784(12):1899-1907 III. Eklund, S.; Lindås, A-C.; Hamnevik, E.; Widersten, M.; Tomkinson, B. Inter-species variation in the pH dependence of tripeptidyl- peptidase II. Manuscript IV. Eklund, S.; Kalbacher, H.; Tomkinson, B. Characterization of the endopeptidase activity of tripeptidyl-peptidase II. Manuscript Paper I and II were published under maiden name (Eriksson). Reprints were made with permission from the respective publishers. Contents Introduction ..................................................................................................... 9 Enzymes ..................................................................................................... 9 Enzymes and pH dependence .............................................................. 11 Peptidases ................................................................................................. 12 Serine peptidases ................................................................................. 14 Intracellular protein
    [Show full text]
  • Aandp2ch25lecture.Pdf
    Chapter 25 Lecture Outline See separate PowerPoint slides for all figures and tables pre- inserted into PowerPoint without notes. Copyright © McGraw-Hill Education. Permission required for reproduction or display. 1 Introduction • Most nutrients we eat cannot be used in existing form – Must be broken down into smaller components before body can make use of them • Digestive system—acts as a disassembly line – To break down nutrients into forms that can be used by the body – To absorb them so they can be distributed to the tissues • Gastroenterology—the study of the digestive tract and the diagnosis and treatment of its disorders 25-2 General Anatomy and Digestive Processes • Expected Learning Outcomes – List the functions and major physiological processes of the digestive system. – Distinguish between mechanical and chemical digestion. – Describe the basic chemical process underlying all chemical digestion, and name the major substrates and products of this process. 25-3 General Anatomy and Digestive Processes (Continued) – List the regions of the digestive tract and the accessory organs of the digestive system. – Identify the layers of the digestive tract and describe its relationship to the peritoneum. – Describe the general neural and chemical controls over digestive function. 25-4 Digestive Function • Digestive system—organ system that processes food, extracts nutrients, and eliminates residue • Five stages of digestion – Ingestion: selective intake of food – Digestion: mechanical and chemical breakdown of food into a form usable by
    [Show full text]
  • Hans Neurath 1909–2002
    Hans Neurath 1909–2002 A Biographical Memoir by Edmond H. Fischer and Earl W. Davie ©2013 National Academy of Sciences. Any opinions expressed in this memoir are those of the authors and do not necessarily reflect the views of the National Academy of Sciences. HANS NEURATH October 29,1909–April 2, 2002 Elected to the NAS, 1961 Hans Neurath, one of the foremost scientists of the 20th century and a man considered one of the founding fathers of modern protein science, passed away on Friday, April 2, 2002. He was the founding chairman of the Department of Biochemistry at the University of Washington School of Medicine in Seattle. After his administrative retirement as department chairman, he became the associate director of research at the Fred Hutchinson Cancer Research Center in Seattle and then, after reaching emeritus status at the University of Washington, he became director of the German Cancer Research Center in Heidelberg, Germany. By Edmond H. Fischer He returned to the University of Washington two years later. and Earl W. Davie Hans was the founding editor of Biochemistry, one of the most prestigious journals in the field and the flagship of some twenty journals published by the American Chemical Society. In 1992, at age 81, he founded Protein Science, a journal of the Protein Society, and he served as its editor-in-chief until 1998. Furthermore, he edited the most comprehensive treatise on proteins, The Proteins. Hans was an avid mountain climber and skier and a first-class pianist. He will be remembered as the person who contributed the most to the field of serine proteases and zymogen activation and, in collaboration with Bert Vallee, to the field of metallo- protein research.
    [Show full text]
  • Proteolytic Cleavage—Mechanisms, Function
    Review Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡ † ‡ Life Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins, resulting in a widespread, irreversible posttranslational modification of the protein’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms
    [Show full text]
  • Following Items. Toluene-Sulfonyl-L-Arginine Methyl
    TRYPTIC ACTIVATION OF ACETYLA TED CHYMOTRYPSINOGEN* BY A. K. BALLS AND C. A. RYAN WESTERN REGIONAL RESEARCH LABORATORY, ALBANY, CALIFORNIAt Communicated December 23, 1963 In a previous paper' we have reported on a rather heavily acetylated a-chymo- trypsinogen that could be activated by trypsin only in the presence of an electrolyte or, alternatively, by the action of unacetylated chymotrypsin prior to, or together with, that of trypsin. The present communication reports further observations on the preparation and composition of such an acetylated zymogen. The condi- tions for -suitable acetylation are different in the presence and in the absence of acetate. After acetylation the solution must be slightly acid, at least for a short time. It also appears that the activation of the acetylated protein in water is pre- vented because some form of acetylated chymotrypsinogen inhibits the action of trypsin on the acetylated zymogen in the absence of salts. This inhibitory ma- terial has been concentrated as a fraction of the mixture that constitutes the product of acetylation. Materials and Methods.-These have been previously described" 2 except for the following items. Trypsin was measured in the same manner as chymotrypsin. The substrate was toluene-sulfonyl-L-arginine methyl ester in 0.001 M tris-maleate buffer' at pH 7.5. The activity of C'4-labeled acetyl was measured in a liquid scintillation spec- trometer. Protein (ca. 0.5 mg) was dissolved in 0.01 N NaOH. The amount of water was limited to 0.2 ml or less in a system containing 15 ml of a fluorescent medium and about an equal volume of finely divided silica.4 Comparison with counts on the CO2 produced by combustion of the protein (which were estimated to be 95% efficient) indicated 48% efficiency in the foregoing system.
    [Show full text]
  • Enzyme Replacement Therapy for Pancreatic Insufficiency
    Enzyme replacement therapy for pancreatic insufficiency: present and future. Aaron Fieker, Jessica Philpott, Martine Armand To cite this version: Aaron Fieker, Jessica Philpott, Martine Armand. Enzyme replacement therapy for pancreatic insuffi- ciency: present and future.. Clinical and Experimental Gastroenterology, Dove Medical Press, 2011, 4 (4), pp.55-73. 10.2147/CEG.S17634. inserm-00701598 HAL Id: inserm-00701598 https://www.hal.inserm.fr/inserm-00701598 Submitted on 25 May 2012 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Clinical and Experimental Gastroenterology Dovepress open access to scientific and medical research Open Access Full Text Article RE VIEW Enzyme replacement therapy for pancreatic kpuwhÞekgpe{<"rtgugpv"cpf"hwvwtg Aaron Fieker1 Abstract: Pancreatic enzyme replacement therapy is currently the mainstay of treatment for Jessica Philpott1 nutrient malabsorption secondary to pancreatic insufficiency. This treatment is safe and has few Martine Armand2 side effects. Data demonstrate efficacy in reducing steatorrhea and fat malabsorption. Effective therapy has been limited by the ability to replicate the physiologic process of enzyme delivery 1Division of Digestive Diseases, University of Oklahoma, OKC, to the appropriate site, in general the duodenum, at the appropriate time. The challenges include OK, USA; 2INSERM, U476 enzyme destruction in the stomach, lack of adequate mixing with the chyme in the duodenum, “Nutrition Humaine et Lipides”, and failing to deliver and activate at the appropriate time.
    [Show full text]
  • A Genomic Analysis of Rat Proteases and Protease Inhibitors
    A genomic analysis of rat proteases and protease inhibitors Xose S. Puente and Carlos López-Otín Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, 33006-Oviedo, Spain Send correspondence to: Carlos López-Otín Departamento de Bioquímica y Biología Molecular Facultad de Medicina, Universidad de Oviedo 33006 Oviedo-SPAIN Tel. 34-985-104201; Fax: 34-985-103564 E-mail: [email protected] Proteases perform fundamental roles in multiple biological processes and are associated with a growing number of pathological conditions that involve abnormal or deficient functions of these enzymes. The availability of the rat genome sequence has opened the possibility to perform a global analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into five catalytic classes: 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. Overall, this distribution is similar to that of the mouse degradome, but significatively more complex than that corresponding to the human degradome composed of 561 proteases and homologs. This increased complexity of the rat protease complement mainly derives from the expansion of several gene families including placental cathepsins, testases, kallikreins and hematopoietic serine proteases, involved in reproductive or immunological functions. These protease families have also evolved differently in the rat and mouse genomes and may contribute to explain some functional differences between these two closely related species. Likewise, genomic analysis of rat protease inhibitors has shown some differences with the mouse protease inhibitor complement and the marked expansion of families of cysteine and serine protease inhibitors in rat and mouse with respect to human.
    [Show full text]
  • Oxidized Phospholipids Regulate Amino Acid Metabolism Through MTHFD2 to Facilitate Nucleotide Release in Endothelial Cells
    ARTICLE DOI: 10.1038/s41467-018-04602-0 OPEN Oxidized phospholipids regulate amino acid metabolism through MTHFD2 to facilitate nucleotide release in endothelial cells Juliane Hitzel1,2, Eunjee Lee3,4, Yi Zhang 3,5,Sofia Iris Bibli2,6, Xiaogang Li7, Sven Zukunft 2,6, Beatrice Pflüger1,2, Jiong Hu2,6, Christoph Schürmann1,2, Andrea Estefania Vasconez1,2, James A. Oo1,2, Adelheid Kratzer8,9, Sandeep Kumar 10, Flávia Rezende1,2, Ivana Josipovic1,2, Dominique Thomas11, Hector Giral8,9, Yannick Schreiber12, Gerd Geisslinger11,12, Christian Fork1,2, Xia Yang13, Fragiska Sigala14, Casey E. Romanoski15, Jens Kroll7, Hanjoong Jo 10, Ulf Landmesser8,9,16, Aldons J. Lusis17, 1234567890():,; Dmitry Namgaladze18, Ingrid Fleming2,6, Matthias S. Leisegang1,2, Jun Zhu 3,4 & Ralf P. Brandes1,2 Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a cau- sal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipo- proteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2- controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.
    [Show full text]
  • Protein-Reactive Natural Products Carmen Drahl, Benjamin F
    Reviews B. F. Cravatt, E. J. Sorensen, and C. Drahl DOI: 10.1002/anie.200500900 Natural Products Chemistry Protein-Reactive Natural Products Carmen Drahl, Benjamin F. Cravatt,* and Erik J. Sorensen* Keywords: enzymes · inhibitors · molecular probes · natural products · structure– activity relationships Angewandte Chemie 5788 www.angewandte.org 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2005, 44, 5788 – 5809 Angewandte Enzyme Inhibitors Chemie Researchers in the post-genome era are confronted with the daunting From the Contents task of assigning structure and function to tens of thousands of encoded proteins. To realize this goal, new technologies are emerging 1. Introduction 5789 for the analysis of protein function on a global scale, such as activity- 2. Natural Products that Target based protein profiling (ABPP), which aims to develop active site- Catalytic Nucleophiles in directed chemical probes for enzyme analysis in whole proteomes. For Enzyme Active Sites 5790 the pursuit of such chemical proteomic technologies, it is helpful to derive inspiration from protein-reactive natural products. Natural 3. Natural Products that Target Non-Nucleophilic Residues in products use a remarkably diverse set of mechanisms to covalently Enzyme Active Sites 5794 modify enzymes from distinct mechanistic classes, thus providing a wellspring of chemical concepts that can be exploited for the design of 4. Targeting Nonenzymatic active-site-directed proteomic probes. Herein, we highlight several Proteins 5802 examples of protein-reactive natural products and illustrate how their 5. Summary and Outlook 5803 mechanisms of action have influenced and continue to shape the progression of chemical proteomic technologies like ABPP. 1.
    [Show full text]