dc.contributor.author
Reimann, Oliver
dc.date.accessioned
2018-06-07T21:27:30Z
dc.date.available
2017-07-10T07:38:07.767Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/7921
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-12120
dc.description
Table of contents Erklärung des Autors ii Declaration iv Publication list v
Talks vii Posters viii Other contributions x Acknowledgement xi Abstract xiii
Kurzzusammenfassung xiv Abbreviations xv Table of contents xxvii 1\.
MOTIVATION 1 2\. INTRODUCTION & BACKGROUND 3 2.1 Amyloid-β, its role in AD and
the amyloid cascade hypothesis 6 2.2 The tau protein 9 2.2.1 Description of
the tau protein 9 2.2.2 Functions of axonal tau 12 2.2.3 Cellular localization
of tau and its function assignments 13 2.2.4 Nuclear tau 14 2.2.5 Tau
phosphorylation and aggregation 16 2.2.6 Hypotheses of tau-related toxicity 21
2.2.7 O-GlcNAcylation of tau 23 2.2.8 Interplay between β-amyloid and tau 26
2.2.9 Diagnosis of Alzheimer’s disease with focus on tau 28 2.2.10 Therapeutic
strategies targeting tau pathology 33 2.3 Peptides and proteins 37 2.3.1 Solid
phase peptide synthesis (SPPS) 38 2.3.2 Introduction of post-translationally
modified amino acids by SPPS 43 2.3.2.1 Phosphopeptides 44 2.3.2.2
Glycopeptides 46 2.3.3 Native chemical ligation (NCL) 47 2.3.4 Methods to
produce C-terminal thioester peptides for linear and convergent peptide and
protein synthesis by NCL reactions 50 2.3.5 N-terminal Cys surrogates and post
ligation desulfurization chemistry 57 2.3.6 The application of new thiol
derivatives in NCL reactions – toward one-pot procedures of NCL and
desulfurization 62 2.3.7 Expressed protein ligation (EPL) 63 2.3.8 Prominent
examples of chemoselective ligations between peptides and proteins other than
NCL and EPL 67 2.3.9 Post-translational modifications introduced by protein
engineering 72 2.4 Tags and cleavable linkers for purification strategies in
chemical biology 75 2.4.1 Introduction 75 2.4.2 Purification tags in chemical
biology 76 2.4.2.1 Tags for type one purification matrix 77 2.4.2.2 Tags for
type two purification matrices 80 2.4.2.3 Tags for purification on type three
matrices 82 2.4.3 Cleavable linker types 82 2.4.3.1 Overview on different
linker types and concepts of their cleavage 82 2.4.3.2 Photocleavable linkers
84 2.4.4 Different applications of cleavable linkers with purification tags 85
2.4.4.1 Hit identification in proteomic research 85 2.4.4.2 Quantitative
chemical proteomics 87 2.4.4.3 Chemical processing of peptides and proteins 88
2.4.4.4 Purification and concomitant labeling of proteins 89 2.4.4.5
Solubility of proteins after expression and purification 92 2.5 Chemical
access to ubiquitinylated proteins 95 3\. OBJECTIVES 96 4\. RESULTS &
DISCUSSION 100 4.1 Tri-phosphorylated C-terminal tau via a native chemical
ligation at an Asn/Val junction 100 4.2 Traceless purification of ligation
products with concomitant desulfurization 125 4.3 Incorporation of Lys
equipped with a photolinker into proteins by amber suppression 162 4.3.1
Introduction to the incorporation of caged amino acids by amber suppression
162 4.3.2 Objective of the project 162 4.3.3 Results and discussion 163 4.3.4
Summary and outlook 166 4.3.5 Responsibility assignment 166 4.4 Semisynthesis
of tag-free O-GlcNAcylated full-length tau by sequential NCL and EPL
reactions, desulfurization and the purification by a photocleavable biotin tag
167 4.5 Toward the semisynthesis of tri-phosphorylated tau proteins and
controls 193 4.5.1 Introduction on previous achievements 193 4.5.2 Outline of
this project 194 4.5.3 Results and discussion 195 4.5.4 Conclusions and
outlook 207 4.5.5 Responsibility assignment 208 4.6 New antibodies against the
tri-phosphorylated PHF-1 epitope of tau 209 4.6.1 Introduction to antibodies
against PHF-1 phosphorylation 209 4.6.2 Outline of this project 211 4.6.3
Results and discussion 211 4.6.3.1 Antibody generation 211 4.6.3.2 Indirect
immunofluorescence experiments with ppp-tau antibodies 213 4.6.3.3 Epitope
mapping 215 4.6.3.4 Evaluation of ppp-tau antibodies by western blot
experiments with semisynthetic tri-phosphorylated tau 218 4.6.3.5 Staining
severe cases of AD and Pick’s Disease 220 4.6.3.6 PHF-1 phosphorylation on tau
in the C.elegans model 222 4.6.3.7 Neuronal model of aging 226 4.6.3.8
Immunoprecipitation (IP) with the 15F1 clone and mass spectrometry of the
pulled out fraction 231 4.6.3.9 ELISA assays with the new antibodies 233 4.6.4
Summary and outlook 241 4.7 Cysteine-functional polymers via thiol-ene
conjugation and subsequent NCL functionalization with peptides 243 4.8
Poly(2-oxazoline)s used for Native Chemical Ligation 280 5\. SUMMARY & OUTLOOK
330 6\. ZUSAMENFASSUNG & AUSBLICK 335 7\. EXPERIMENTAL PART 340 7.1 Exp:
Materials and methods 340 7.2 Experimental details on: Incorporation of the
PCB-Lys into proteins by amber suppression 342 7.2.1 Exp: Synthesis of the PC-
Lys building block by a solid phase approach 342 7.2.2 Exp: Synthesis of the
PC-Lys building block by in-solution approach 343 7.2.3 Exp: Characterization
of GFP containing the unnatural amino acid by mass spectrometry 346 7.3
Experimental details on: Toward the semisynthesis of the tri-phosphorylated
tau proteins and controls 349 7.3.1 Exp: Synthesis of the tri-phosphorylated
N-peptide 5 for NCL 349 7.3.2 Exp: One-pot native chemical ligation and
desulfurization toward tri-phosphorylated tau peptide (7) using TFET as thiol
additive 351 7.3.3 Exp: Semisynthesis of unlabeled tri-phosphorylated tau (9)
352 7.3.4 Exp: Characterization of semisynthetic protein 9 353 7.3.5 Exp:
Semisynthesis of unphosphorylated, partially 15N-labeled tau (12) 354 7.3.6
Exp: Characterization of semisynthetic unphosphorylated and 15N-labeled tau
(12) 354 7.3.7 Exp: Expression of 15N-labeled tau[1-389]-intein-GST fusion-
protein 355 7.3.8 Exp: Synthesis of tau[390-441] (15) 356 7.3.9 Exp: EPL with
unbiotinylated peptide 15 and subsequent HPLC purification 357 7.3.10 Exp: EPL
of 7 and 16 toward double-labeled tri-phosphorlylated tau (17) 358 7.3.11 Exp:
Experiments on tau aggregation during EPL 358 7.4 Experimental details on: New
antibodies against the tri-phosphorylated PHF-1 epitope of tau 360 7.4.1 Exp:
Antibody generation 360 Synthesis of peptide 18 360 7.4.2 Exp: Epitope mapping
361 7.4.3 Exp: Evaluation of antibody clones by semisynthetic tri-
phosphorylated tau proteins by western blot experiments 364 7.4.4 Exp: PHF-1
phosphorylation on tau in the C.elegans model 365 7.4.5 Exp:
Immunoprecipitation (IP) with the 15F1 clone and mass spectrometry of the
pulled-out fraction 366 7.4.6 Exp: ELISA assays with the new antibodies 367
7.4.6.1 Identification of the most potent clone (according to setting shown in
Figure 50) 367 7.4.6.2 Measurement of total tau content by ELISA assays of a
small cohort with the INNOTEST hTau Ag kit and comparison to phospho-tau
levels detected with the 15F1 antibody 370 7.4.6.3 General Protocol for ELISA
assays shown in Figure 51 371 7.4.6.4 Cross-reactivity study 372 8\.
REFERENCES 376 9\. CURRICULUM VITAE 396
dc.description.abstract
The work of this Ph.D. puts the focus on tau, a key protein in Alzheimer’s
Disease (AD). During progression of AD, tau becomes abnormally phosphorylated.
This high degree of phosphorylation is associated to key events in the
disease, such as protein aggregation and destabilization of the neuronal
integrity. A natural regulator of tau phosphorylation is O-GlcNAcylation, a
post-translational modification, placed on the same residues as phosphate.
Here, new synthetic pathways to generate homogeneously phosphorylated or
O-GlcNAcylated tau proteins are presented, which are accomplished by combined
approaches of native chemical ligation (NCL) and expressed protein ligation
(EPL). A new method was established, in which purification was achieved after
ligation and desulfurization by means of a traceless photocleavable biotin tag
that was installed during solid phase peptide synthesis (SPPS) in synthetic
tau fragments. This protocol allowed access to tag-free tau peptides and
proteins, which were conveniently desulfurized post-ligation during peptide
immobilization, if required. The molecular targets were either phosphorylated
or O-GlcNAcylated tau proteins, carrying these post-translational
modifications in the AD relevant paired helical filament 1 (PHF-1) epitope
(Ser396/400/404). To get more insights into the impact of tau phosphorylation
in PHF-1 in cellulo and in vivo, monoclonal antibodies against tau were
generated, tri-phosphorylated in this particular epitope. The antibodies were
characterized by several experimental settings and enabled the generation of
new insights into cellular tau localization. Moreover, the exploration of the
diagnostic potential of these new antibody clones was started.
de
dc.description.abstract
Der Fokus dieser Dissertation liegt auf Tau, einem Schlüsselprotein in der
Alzheimer Erkrankung. Im Fortlauf von Alzheimer wird Tau hyperphosphoryliert,
wobei der hohe Grad der Phosphorylierung mit Schlüsselereignissen der
Krankheit wie der Aggregation von Tau oder dem Verlust der neuronalen
Integrität in Verbindung gebracht wird. Die post-translationale Modifizierung
der O-GlcNAcylierung wird ebenfalls an Tau beobachtet und gilt als natürlicher
Regulator der Phosphorylierung. Hier werden neue Syntheserouten präsentiert,
die den Zugang zu homogen-phosphorylierten und O-GlcNAcylierten Tau Derivaten
durch kombinatorische Ansätze aus nativer chemischer Ligation (NCL) und
exprimierter Protein Ligation (EPL) ermöglichen. Es wurde eine neue Methode
entwickelt, die eine Reinigung von Ligationsprodukten durch einen spurlos
photospaltbaren Biotin-Tag ermöglichten, der während der Festphasen Peptid
Synthese in die synthetischen Peptid-Fragmente eingeführt werden kann. Dieses
neuartige Protokoll ermöglichte den synthetischen Zugang zu Tag-freien Tau
Proteinen, die ohne großen experimentellen Aufwand während ihrer
Immobilisierung entschwefelt werden konnten. Die molekularen Ziele dieser
Arbeit waren phosphorylierte oder O-GlcNAcylierte Tau Proteine, die diese
post-translationalen Modifikationen im sog. „paired helical filament“ 1
(PHF-1) Epitop (Ser396/400/404) enthalten. Um weiteres Wissen über den
Einfluss starker Tau Phosphorylierung in PHF-1 in cellulo und in vivo zu
erhalten wurden monoklonale Antikörper gegen dreifach phosphoryliertes Tau
entwickelt und diese dann in unterschiedlichen Experimenten charakterisiert.
So konnten neue Einsichten über die zelluläre Lokalisation von Tau in
Abhängigkeit von der PHF-1 Phosphorylierung gesammelt werden. Darüber hinaus
wurde begonnen, das diagnostische Potential der neuen Antikörper Klone zu
erforschen.
de
dc.format.extent
xxvii, 398 Seiten
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject
alzheimer's disease
dc.subject
protein chemistry
dc.subject.ddc
500 Naturwissenschaften und Mathematik::540 Chemie::547 Organische Chemie
dc.title
Tag-free semisynthetic tau proteins and novel antibodies targeted against
phospho-tau
dc.contributor.contact
oliver.reimann@enviropep.de
dc.contributor.firstReferee
Prof. Dr. Christian P. R. Hackenberger
dc.contributor.furtherReferee
Prof. Dr. Beate Koksch
dc.date.accepted
2016-11-15
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000104575-2
dc.title.translated
Tag-freie semisynthetische Tau Proteine und neue Antikörper gegen Phospho-Tau
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000104575
refubium.mycore.derivateId
FUDISS_derivate_000000021786
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access