DOCSLIB.ORG

And Β-Secretase in Alzheimer's Disease

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
And Β-Secretase in Alzheimer's Disease Deutsches Zentrum für Neurodegenerative Erkrankungen Standort München α- and β-secretase in Alzheimer’s disease: Identity and physiological substrates Peer-Hendrik Kuhn Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doctor of Philosophy (Ph.D.) genehmigten Dissertation. Vorsitzender: apl. Prof. Dr. Helmuth K. H. Adelsberger Prüfer der Dissertation: 1. Univ.- Prof. Dr. Thomas Misgeld 2. Priv.-Doz. Dr. Stefan Lichtenthaler Die Dissertation wurde am 29.2.2012 bei der Fakultät für Medizin der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 08.03.2012 angenommen. Content Content ........................................................................................................................1 Table index .................................................................................................................. 3 1 Introduction ...................................................................................................... 4 1.1 Alzheimer’s disease ........................................................................................... 4 1.1.1 History ................................................................................................................ 4 1.1.2 Epidemiology ...................................................................................................... 5 1.1.3 Histopathology of Alzheimer’s disease ............................................................... 5 1.1.4 Familial Alzheimer’s disease (FAD) .................................................................... 6 1.1.5 Sporadic Alzheimer’s disease ............................................................................. 7 1.1.6 The Amyloid Cascade Hypothesis ...................................................................... 9 1.2 Proteases ......................................................................................................... 11 1.2.1 Regulated intramembrane proteolysis (RIP) ..................................................... 12 1.2.2 Ectodomain shedding ....................................................................................... 13 1.2.3 Intramembrane Proteolysis ............................................................................... 16 1.2.4 The Amyloid Precursor Protein (APP) .............................................................. 18 1.2.5 Proteolytic Processing of the Amyloid Precursor Protein .................................. 19 1.2.6 Therapeutic strategies of Alzheimer’s disease based on protease inhibition or modulation ................................................................................................... 20 1.3 Click chemistry – Bioorthogonal reactions enable purification of subproteomes or investigation of posttranslational modifications ...................... 22 1.3.1 Bioorthogonal-Definition ................................................................................... 22 1.3.2 Click Chemistry reactions ................................................................................. 22 1.3.3 Click chemistry applications in molecular biology ............................................. 24 1.4 Goal of this work ............................................................................................... 25 2 Material and Methods ..................................................................................... 27 2.1 DNA methods ................................................................................................... 27 2.1.1 Polymerase Chain reaction (PCR) .................................................................... 27 2.1.2 Restriction digest .............................................................................................. 28 2.1.3 Cloned and used DNA constructs and list of oligonucleotides .......................... 29 2.1.4 Separation of DNA fragments ........................................................................... 30 2.1.5 Purification of DNA fragments out of agarose slices ......................................... 31 2.1.6 DNA ligation ..................................................................................................... 31 2.1.7 Production of chemical competent E.coli bacteria ............................................. 32 2.1.8 Transformation of chemical competent bacteria ............................................... 33 2.1.9 Small scale purification of plasmid DNA ........................................................... 34 2.1.10 Large scale purification of plasmid DNA ........................................................... 35 2.1.11 Sequence verification of newly cloned DNA constructs .................................... 35 2.2 Cell culture methods ......................................................................................... 35 2.2.1 Culture and manipulation of immortalized cell lines .......................................... 35 2.2.2 Lentivirus production ........................................................................................ 38 2.2.3 Isolation and culture of primary cortical neurons ............................................... 39 2.2.4 Lentiviral infection of primary cortical neurons .................................................. 41 2.2.5 Compound treatment of cells including primary neurons .................................. 41 2.3 Protein analysis methods ................................................................................. 41 Chapter 1 Content 2 2.3.1 Cell lysis ........................................................................................................... 41 2.3.2 Extraction of soluble and insoluble fraction of brain tissue ................................ 42 2.3.3 Protein measurement ....................................................................................... 43 2.3.4 SDS gel electrophoresis ................................................................................... 44 2.3.5 Separation of proteins with low molecular weight ............................................. 45 2.3.6 Coomassie staining of SDS gels ...................................................................... 46 2.3.7 Western Blot ..................................................................................................... 47 2.3.8 Antibody list ...................................................................................................... 48 2.3.9 Detection and Quantification............................................................................. 49 2.3.10 Analysis of immunoprecipitated peptides with matrix-assisted laser desorption ionization (MALDI) mass spectrometry ............................................ 49 2.3.11 Secretome protein enrichment with click sugars (SPECS) ................................ 50 2.3.12 Preparation of peptides for mass spectrometric measurement ......................... 51 2.3.13 Online HPLC and measurement of peptides with an LTQ-Velos-Orbitrap ......... 52 2.3.14 Analysis of mass spectrometric raw data with the MaxQuant Suite .................. 53 2.3.15 Construction of protein filters using SWISS PROTdatabases in excel ............. 53 2.4 Methods for RNA analysis ................................................................................ 53 2.4.1 RNA extraction ................................................................................................. 53 2.4.2 Reverse transcription........................................................................................ 54 2.4.3 qRT-PCR .......................................................................................................... 54 3 Results ............................................................................................................ 55 3.1 Construction of potent lentiviral shRNA and lentiviral overexpression vectors .. 55 3.2 ADAM10 is the physiological α-secretase in primary cortical neurons .............. 61 3.3 A Cre-mediated conditional knockout of ADAM10 independently confirms ADAM10 as the sole APP alpha secretase in primary cortical neurons ............ 63 3.4 ADAM10 cleaves between Lysine 16 and Leucine 17 of the Amyloid β domain of APP ................................................................................................. 64 3.5 Establishment of the method Secretome Protein Enrichment with Click Sugars - SPECS ............................................................................................... 67 3.6 Determination of the HEK293T and the neuronal secretome ............................ 70 3.7 Identification of the BACE1 sheddome in primary cortical neurons with the SPECS method ................................................................................................ 72 3.8 Validation of CHL1, L1CAM, Sez6 and Contactin-2 as new BACE1 substrates by Western Blot analysis ................................................................. 74 4 Discussion ...................................................................................................... 79 4.1.1 Modified lentiviral vectors allow knockdown and overexpression of membrane proteins .......................................................................................... 80 4.1.2 ADAM10 is the physiologically relevant alpha secretase of APP ...................... 82 4.1.3 Secretome Protein enrichment with click sugars enables identification of novel BACE1 substrates in primary cortical neurons ........................................ 86
Load more

Recommended publications
  • Supplemental Figure 1. Vimentin
    Double mutant specific genes Transcript gene_assignment Gene Symbol RefSeq FDR Fold- FDR Fold- FDR Fold- ID (single vs. Change (double Change (double Change wt) (single vs. wt) (double vs. single) (double vs. wt) vs. wt) vs. single) 10485013 BC085239 // 1110051M20Rik // RIKEN cDNA 1110051M20 gene // 2 E1 // 228356 /// NM 1110051M20Ri BC085239 0.164013 -1.38517 0.0345128 -2.24228 0.154535 -1.61877 k 10358717 NM_197990 // 1700025G04Rik // RIKEN cDNA 1700025G04 gene // 1 G2 // 69399 /// BC 1700025G04Rik NM_197990 0.142593 -1.37878 0.0212926 -3.13385 0.093068 -2.27291 10358713 NM_197990 // 1700025G04Rik // RIKEN cDNA 1700025G04 gene // 1 G2 // 69399 1700025G04Rik NM_197990 0.0655213 -1.71563 0.0222468 -2.32498 0.166843 -1.35517 10481312 NM_027283 // 1700026L06Rik // RIKEN cDNA 1700026L06 gene // 2 A3 // 69987 /// EN 1700026L06Rik NM_027283 0.0503754 -1.46385 0.0140999 -2.19537 0.0825609 -1.49972 10351465 BC150846 // 1700084C01Rik // RIKEN cDNA 1700084C01 gene // 1 H3 // 78465 /// NM_ 1700084C01Rik BC150846 0.107391 -1.5916 0.0385418 -2.05801 0.295457 -1.29305 10569654 AK007416 // 1810010D01Rik // RIKEN cDNA 1810010D01 gene // 7 F5 // 381935 /// XR 1810010D01Rik AK007416 0.145576 1.69432 0.0476957 2.51662 0.288571 1.48533 10508883 NM_001083916 // 1810019J16Rik // RIKEN cDNA 1810019J16 gene // 4 D2.3 // 69073 / 1810019J16Rik NM_001083916 0.0533206 1.57139 0.0145433 2.56417 0.0836674 1.63179 10585282 ENSMUST00000050829 // 2010007H06Rik // RIKEN cDNA 2010007H06 gene // --- // 6984 2010007H06Rik ENSMUST00000050829 0.129914 -1.71998 0.0434862 -2.51672
    [Show full text]
  • Supplementary Data
    Supplemental Data A novel mouse model of X-linked nephrogenic diabetes insipidus: Phenotypic analysis and therapeutic implications Jian Hua Li, Chung-Lin Chou, Bo Li, Oksana Gavrilova, Christoph Eisner, Jürgen Schnermann, Stasia A. Anderson, Chu-Xia Deng, Mark A. Knepper, and Jürgen Wess Supplemental Methods Metabolic cage studies. Animals were maintained in mouse metabolic cages (Hatteras Instruments, Cary, NC) under controlled temperature and light conditions (12 hr light and dark cycles). Mice received a fixed daily ration of 6.5 g of gelled diet per 20 g of body weight per day. The gelled diet was composed of 4 g of Basal Diet 5755 (Test Diet, Richmond, IN), 2.5 ml of deionized water, and 65 mg agar. Preweighted drinking water was provided ad libitum during the course of the study. Mice were acclimated in the metabolic cages for 1-2 days. Urine was collected under mineral oil in preweighted collection vials for successive 24 hr periods. Analysis of GPCR expression in mouse IMCD cells via TaqMan real-time qRT-PCR. Total RNA prepared from mouse IMCD tubule suspensions was reverse transcribed as described under Experimental Procedures. Tissues from ten 10-week old C57BL/6 WT mice were collected and pooled for each individual experiment. cDNA derived from 640 ng of RNA was mixed with an equal volume of TaqMan gene expression 2 x master mix (Applied Biosystems, Foster City, CA). 100 μl-aliquots of this mixture (corresponding to 80 ng of RNA) were added to each of the 8 fill ports of a 384-well plate of a mouse GPCR array panel (Applied Biosystems).
    [Show full text]
  • Monitoring Nociception by Analyzing Gene Expression Changes in the Central Nervous System of Mice
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2010 Monitoring nociception by analyzing gene expression changes in the central nervous system of mice Asner, I N Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-46678 Dissertation Originally published at: Asner, I N. Monitoring nociception by analyzing gene expression changes in the central nervous system of mice. 2010, University of Zurich, Vetsuisse Faculty. Monitoring Nociception by Analyzing Gene Expression Changes in the Central Nervous System of Mice Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Igor Asner von St. Cergue VD Promotionskomitee Prof. Dr. Peter Sonderegger Prof. Dr. Kurt Bürki Prof. Dr. Hanns Ulrich Zeilhofer Dr. Paolo Cinelli (Leitung der Dissertation) Zürich, 2010 Table of contents Table of content Curriculum vitae 6 Publications 9 Summary 11 Zusammenfassung 14 1. Introduction 17 1.1. Pain and nociception 17 1.1.1 Nociceptive neurons and Mechanoceptors 18 1.1.2 Activation of the nociceptive neurons at the periphery 21 1.1.2.1 Response to noxious heat 22 1.1.2.2 Response to noxious cold 23 1.1.2.3 Response to mechanical stress 24 1.1.3 Nociceptive message processing in the Spinal Cord 25 1.1.3.1 The lamina I and the ascending pathways 25 1.1.3.2 The lamina II and the descending pathways 26 1.1.4 Pain processing and integration in the brain 27 1.1.4.1 The Pain Matrix 27 1.1.4.2 Activation of the descending pathways 29 1.1.5 Inflammatory Pain 31 1.2.
    [Show full text]
  • Identification of New Substrates and Physiological Relevance
    Université de Montréal The Multifaceted Proprotein Convertases PC7 and Furin: Identification of New Substrates and Physiological Relevance Par Stéphanie Duval Biologie Moléculaire, Faculté de médecine Thèse présentée en vue de l’obtention du grade de Philosophiae doctor (Ph.D) en Biologie moléculaire, option médecine cellulaire et moléculaire Avril 2020 © Stéphanie Duval, 2020 Résumé Les proprotéines convertases (PCs) sont responsables de la maturation de plusieurs protéines précurseurs et sont impliquées dans divers processus biologiques importants. Durant les 30 dernières années, plusieurs études sur les PCs se sont traduites en succès cliniques, toutefois les fonctions spécifiques de PC7 demeurent obscures. Afin de comprendre PC7 et d’identifier de nouveaux substrats, nous avons généré une analyse protéomique des protéines sécrétées dans les cellules HuH7. Cette analyse nous a permis d’identifier deux protéines transmembranaires de fonctions inconnues: CASC4 et GPP130/GOLIM4. Au cours de cette thèse, nous nous sommes aussi intéressé au rôle de PC7 dans les troubles comportementaux, grâce à un substrat connu, BDNF. Dans le chapitre premier, je présenterai une revue de la littérature portant entre autres sur les PCs. Dans le chapitre II, l’étude de CASC4 nous a permis de démontrer que cette protéine est clivée au site KR66↓NS par PC7 et Furin dans des compartiments cellulaires acides. Comme CASC4 a été rapporté dans des études de cancer du sein, nous avons généré des cellules MDA- MB-231 exprimant CASC4 de type sauvage et avons démontré une diminution significative de la migration et de l’invasion cellulaire. Ce phénotype est causé notamment par une augmentation du nombre de complexes d’adhésion focale et peut être contrecarré par la surexpression d’une protéine CASC4 mutante ayant un site de clivage optimale par PC7/Furin ou encore en exprimant une protéine contenant uniquement le domaine clivé N-terminal.
    [Show full text]
  • ADGRE2-NTF Is Regulated by Site- Specific N-Glycosylation
    www.nature.com/scientificreports OPEN Membrane-association of EMR2/ ADGRE2-NTF is regulated by site- specifc N-glycosylation Received: 19 December 2017 Yi-Shu Huang1,4, Nien-Yi Chiang1, Gin-Wen Chang1 & Hsi-Hsien Lin1,2,3 Accepted: 27 February 2018 The evolutionarily conserved adhesion G protein-coupled receptors (aGPCRs) play critical roles in Published: xx xx xxxx biological processes as diverse as brain development, cell polarity and innate immune functions. A defning feature of aGPCRs is the GPCR autoproteolysis inducing (GAIN) domain capable of self- catalytic cleavage, resulting in the generation of an extracellular N-terminal fragment (NTF) and a seven-transmembrane C-terminal fragment (CTF) involved in the cellular adhesion and signaling functions, respectively. Interestingly, two diferent NTF subtypes have previously been identifed, namely an NTF that couples non-covalently with the CTF and a membrane-associated NTF that tethers on cell surface independently. The two NTF subtypes are expected to regulate aGPCR signaling via distinct mechanisms however their molecular characteristics are largely unknown. Herein, the membrane-associated NTF of EMR2/ADGRE2 is investigated and found to be modifed by diferential N-glycosylation. The membrane association of EMR2-NTF occurs in post-ER compartments and site- specifc N-glycosylation in the GAIN domain is involved in modulating its membrane-association ability. Finally, a unique amphipathic α-helix in the GAIN domain is identifed as a putative membrane anchor of EMR2-NTF. These results provide novel insights into the complex interaction and activation mechanisms of aGPCRs. Characterized by a long extracellular domain (ECD) with cell-adhesion functions and a seven-transmembrane (7TM) domain with signaling functions, the adhesion G protein-coupled receptors (aGPCRs) have been impli- cated in diverse biological activities and human diseases1.
    [Show full text]
  • Article Reference
    Article Toxoplasma gondii transmembrane microneme proteins and their modular design SHEINER, Lilach, et al. Abstract Summary Host cell invasion by the Apicomplexa critically relies on regulated secretion of transmembrane micronemal proteins (TM-MICs). Toxoplasma gondii possesses functionally non-redundant MICs complexes that participate in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. The TM-MICs are released onto the parasite's surface as complexes capable of interacting with host cell receptors. Additionally, TgMIC2 simultaneously connects to the actomyosin system via binding to aldolase. During invasion these adhesive complexes are shed from the surface notably via intramembrane cleavage of the TM-MICs by a rhomboid protease. Some TM-MICs act as escorters and assure trafficking of the complexes to the micronemes. We have investigated the properties of TgMIC6, TgMIC8, TgMIC8.2, TgAMA1 and the new micronemal protein TgMIC16 with respect to interaction with aldolase, susceptibility to rhomboid cleavage and presence of trafficking signals. We conclude that several TM-MICs lack targeting information within their C-terminal domains, indicating that trafficking depends on yet unidentified [...] Reference SHEINER, Lilach, et al. Toxoplasma gondii transmembrane microneme proteins and their modular design. Molecular microbiology, 2010, vol. 77, no. 4, p. 912-929 DOI : 10.1111/j.1365-2958.2010.07255.x PMID : 20545864 Available at: http://archive-ouverte.unige.ch/unige:12352 Disclaimer: layout of this document may differ from the published version. 1 / 1 Molecular Microbiology (2010) ᭿ doi:10.1111/j.1365-2958.2010.07255.x Toxoplasma gondii transmembrane microneme proteins and their modular designmmi_7255 1..18 Lilach Sheiner,1†‡ Joana M.
    [Show full text]
  • An Entamoeba Histolytica Rhomboid Protease with Atypical Specificity Cleaves a Surface Lectin Involved in Phagocytosis and Immune Evasion
    Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press An Entamoeba histolytica rhomboid protease with atypical specificity cleaves a surface lectin involved in phagocytosis and immune evasion Leigh A. Baxt,1 Rosanna P. Baker,2 Upinder Singh,1,4 and Sinisa Urban2,3 1Departments of Internal Medicine and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA; 2Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA Rhomboid proteases are membrane-embedded enzymes conserved in all kingdoms of life, but their cellular functions across evolution are largely unknown. Prior work has uncovered a role for rhomboid enzymes in host cell invasion by malaria and related intracellular parasites, but this is unlikely to be a widespread function, even in pathogens, since rhomboid proteases are also conserved in unrelated protozoa that maintain an extracellular existence. We examined rhomboid function in Entamoeba histolytica, an extracellular, parasitic ameba that is second only to malaria in medical burden globally. Despite its large genome, E. histolytica encodes only one rhomboid (EhROM1) with residues necessary for protease activity. EhROM1 displayed atypical substrate specificity, being able to cleave Plasmodium adhesins but not the canonical substrate Drosophila Spitz. We searched for substrates encoded in the ameba genome and found EhROM1 was able to cleave a cell surface lectin specifically. In E. histolytica trophozoites, EhROM1 changed localization to vesicles during phagocytosis and to the posterior cap structure during surface receptor shedding for immune evasion, in both cases colocalizing with lectins. Collectively these results implicate rhomboid proteases for the first time in immune evasion and suggest that a common function of rhomboid enzymes in widely divergent protozoan pathogens is to break down adhesion proteins.
    [Show full text]
  • Substrates and Physiological Functions of Secretase Rhomboid Proteases
    G Model YSCDB-2100; No. of Pages 9 ARTICLE IN PRESS Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx Contents lists available at ScienceDirect Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb Substrates and physiological functions of secretase rhomboid proteases ∗ Viorica L. Lastun, Adam G. Grieve, Matthew Freeman Dunn School of Pathology, South Parks Road, Oxford, OX1 3RE, United Kingdom a r t i c l e i n f o a b s t r a c t Article history: Rhomboids are conserved intramembrane serine proteases with widespread functions. They were the Received 3 May 2016 earliest discovered members of the wider rhomboid-like superfamily of proteases and pseudoproteases. Received in revised form 26 July 2016 The secretase class of rhomboid proteases, distributed through the secretory pathway, are the most Accepted 31 July 2016 numerous in eukaryotes, but our knowledge of them is limited. Here we aim to summarise all that has Available online xxx been published on secretase rhomboids in a concise encyclopaedia of the enzymes, their substrates, and their biological roles. We also discuss emerging themes of how these important enzymes are regulated. © 2016 Published by Elsevier Ltd. Contents 1. Introduction . 00 2. Rhomboids – general principles . 00 3. Eukaryotic secretase rhomboids . 00 3.1. Drosophila rhomboids . 00 3.1.1. Rhomboid-1 . 00 3.1.2. Rhomboid-2 . 00 3.1.3. Rhomboid-3 . 00 3.1.4. Rhomboid-4 . 00 3.1.5. Rhomboid-6 . 00 3.2. Mammalian rhomboids . 00 3.2.1. RHBDL1 . 00 3.2.2. RHBDL2 . 00 3.2.3.
    [Show full text]
  • New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation
    International Journal of Molecular Sciences Review New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation Sandra Berndt * and Ines Liebscher Rudolf Schönheimer Institute of Biochemistry, Molecular Biochemistry, Medical Faculty, University of Leipzig, 04103 Leipzig, Germany; [email protected] * Correspondence: [email protected]; Tel.: +49-341-9722175 Abstract: Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)- mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much Citation: Berndt, S.; Liebscher, I. is known of their contribution to different signaling pathways.
    [Show full text]
  • The Rhomboid Protease Family: a Decade of Progress on Function and Mechanism Sinisa Urban* and Seth W Dickey
    Urban and Dickey Genome Biology 2011, 12:231 http://genomebiology.com/2011/12/10/231 REVIEW The rhomboid protease family: a decade of progress on function and mechanism Sinisa Urban* and Seth W Dickey Summary intramembrane proteolysis, rhomboid enzymes are the only family that were not discovered from the direct Rhomboid proteases are the largest family of study of human disease. e name ‘rhomboid’ has its enzymes that hydrolyze peptide bonds within the cell origin deep in the rich folklore of Drosophila genetics. membrane. Although discovered to be serine proteases Rhomboid emerged from the historic quest to identify all only a decade ago, rhomboid proteases are already genes required to organize construction of a free-living considered to be the best understood intramembrane organism from a single cell [8,9]. Because genes were proteases. The presence of rhomboid proteins in all named after the altered appearance of the mutant larval domains of life emphasizes their importance but cuticle, the mis-shaped, rhombus-like head skeleton of makes their evolutionary history dicult to chart with the mutant embryo earned rhomboid its name. Mutating condence. Phylogenetics nevertheless oers three the growth factor that rhomboid activates yielded guiding principles for interpreting rhomboid function. indistinguishable head-skeleton defects, and was named The near ubiquity of rhomboid proteases across spitz (‘pointed’ in German). evolution suggests broad, organizational roles that are e rhomboid gene was cloned and sequenced by Bier not directly essential for cell survival. Functions have and colleagues in 1990, revealing a seven transmembrane been deciphered in only about a dozen organisms and (7TM) protein with no homology to any sequence known fall into four general categories: initiating cell signaling at the time [10].
    [Show full text]
  • Activity-Based Profiling of Proteases
    BI83CH11-Bogyo ARI 3 May 2014 11:12 Activity-Based Profiling of Proteases Laura E. Sanman1 and Matthew Bogyo1,2,3 Departments of 1Chemical and Systems Biology, 2Microbiology and Immunology, and 3Pathology, Stanford University School of Medicine, Stanford, California 94305-5324; email: [email protected] Annu. Rev. Biochem. 2014. 83:249–73 Keywords The Annual Review of Biochemistry is online at biochem.annualreviews.org activity-based probes, proteomics, mass spectrometry, affinity handle, fluorescent imaging This article’s doi: 10.1146/annurev-biochem-060713-035352 Abstract Copyright c 2014 by Annual Reviews. All rights reserved Proteolytic enzymes are key signaling molecules in both normal physi- Annu. Rev. Biochem. 2014.83:249-273. Downloaded from www.annualreviews.org ological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA by Stanford University - Main Campus Lane Medical Library on 08/28/14. For personal use only. and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the ap- plication of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers. 249 BI83CH11-Bogyo ARI 3 May 2014 11:12 gens that contain inhibitory prodomains that Contents must be removed for the protease to become active.
    [Show full text]
  • Plastid Intramembrane Proteolysis☆
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1847 (2015) 910–914 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbabio Review Plastid intramembrane proteolysis☆ Zach Adam ⁎ The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel article info abstract Article history: Progress in the field of regulated intramembrane proteolysis (RIP) in recent years has not surpassed plant Received 22 October 2014 biology. Nevertheless, reports on RIP in plants, and especially in chloroplasts, are still scarce. Of the four different Received in revised form 9 December 2014 families of intramembrane proteases, only two have been linked to chloroplasts so far, rhomboids and site-2 Accepted 12 December 2014 proteases (S2Ps). The lack of chloroplast-located rhomboid proteases was associated with reduced fertility and Available online 18 December 2014 aberrations in flower morphology, probably due to perturbations in jasmonic acid biosynthesis, which occurs fi Keywords: in chloroplasts. Mutations in homologues of S2P resulted in chlorophyll de ciency and impaired chloroplast Chloroplast development, through a yet unknown mechanism. To date, the only known substrate of RIP in chloroplasts is a Protease PHD transcription factor, located in the envelope. Upon proteolytic cleavage by an unknown protease, the soluble Rhomboid protease N-terminal domain of this protein is released from the membrane and relocates to the nucleus, where it activates Site-2 protease the transcription of the ABA response gene ABI4.
    [Show full text]