Verfügbarkeit: Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens

Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens:

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Bibliographische Detailangaben
Beteilige Person:	Stein, Martin Lorenz (VerfasserIn)
Format:	Hochschulschrift/Dissertation Buch
Sprache:	Englisch
Veröffentlicht:	2013
Schlagwörter:	Hochschulschrift
Links:	http://mediatum.ub.tum.de/node?id=1177664 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20131216-1177664-0-6 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027003422&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
Umfang:	99 S. Ill., graph. Darst.

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Datensatz im Suchindex

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adam_text	TABLE OF CONTENTS TABLE OF CONTENTS 1. SUMMARY 8 2. ZUSAMMENFASSUNG 9 3. INTRODUCTION 12 3.1 THE UBIQUITIN-PROTEASOME-SYSTEM 12 3.1.1 UBIQUITINYLATION 12 3.1.2 THE 26S PROTEASOME 14 3.1.3 THE CORE PARTICLE 15 3.2 PRINCIPLES AND APPLICATIONS OF SYNTHETIC PROTEASOME INHIBITORS 19 3.3 NATURAL PROTEASOME INHIBITORS 23 3.4 PROTEASOME INHIBITORS AS VIRULENCE FACTORS FROM PATHOGENIC ORIGIN 25 3.5 OVERVIEW OF CURRENTLY APPLIED METHODS FOR PROTEASOMAL INHIBITOR DETECTION 28 4. OBJECTIVE 32 5. MATERIALS AND METHODS 34 5.1 MATERIALS 34 5.1.1 CHEMICALS 34 5.1.2 GROWTH MEDIA 34 5.1.3 STRAINS 35 5.1.4 FLUOROGENIC SUBSTRATES 36 5.1.5 INSTRUMENTS 36 5.1.6 COMPUTER SOFTWARE AND BIOINFORMATIC TOOLS 39 5.2 GENERATION OF PEPTIDE SUBSTRATES 39 5.2.1 SOLID PHASE SYNTHESIS 39 5.2.2 CLEAVAGE REACTION AND PURIFICATION 40 5.3 PROTEIN CHEMISTRY 41 5.3.1 PURIFICATION OF THE 20S PROTEASOME 41 5.3.2 SDS-PAGE ANALYSIS 42 5 HTTP://D-NB.INFO/1048244091 TABLE OF CONTENTS 5.3.3 DETERMINATION OF PROTEIN CONCENTRATIONS 43 5.4 ACTIVITY ASSAYS 44 5.4.1 ARTIFICIAL FLUORESCENT PROBES AND IC50 MEASUREMENTS 44 5.4.2 NATIVE GEL FLUORESCENCE ANALYSIS 44 5.4.3 SDS-PAGE SHIFT ASSAY 45 5.4.4 PEPTIDE DIGESTION AND PRODUCT CLEAVAGE PATTERN ANALYSIS 46 5.4.5 NMR ANALYSIS OF LABELED PEPTIDE SUBSTRATES 47 5.5 ISOLATION OF NATURAL PRODUCTS 48 5.5.1 SCREENING OF GROWTH CONDITIONS 48 5.5.2 SECRETION AND PURIFICATION OF THE SYRBACTINS CEPI AND GLBA 49 5.5.3 LC-MS, ID- AND 2D-NMR STRUCTURE ELUCIDATION 50 5.6 PROTEIN CRYSTALLOGRAPHY 50 5.6.1 CRYSTALLIZATION OF THE YEAST 20S PROTEASOME 50 5.6.2 DATA COLLECTION, PROCESSING AND STRUCTURE DETERMINATION 51 5.7 CELL CULTURE EXPERIMENTS 51 5.7.1 CELL VIABILITY ASSAYS AND KINETIC EXPERIMENTS 51 5.7.2 NF-KB PATHWAY ANALYSIS 52 6. RESULTS 53 6.1 DEVELOPMENT OF A NMR-BASED PROTEASOME ASSAY 53 6.1.1 PEPTIDE DESIGN AND DIGESTION ANALYSES 53 6.1.2 NMR ANALYSIS OF THE CLEAVAGE REACTION 55 6.1.3 PRIMARY SEQUENCE OPTIMIZATION 56 6.2 ASSAY VERIFICATION IN A REAL-CASE SCENARIO 58 6.2.1 DETECTION OF SYLA IN CULTURES OF P. SYRINGAE BY ORTHOGONAL METHODOLOGIES 58 6.2.2 ANALYSIS OF SYLA CONCENTRATIONS BY NMR ASSAYS 59 6.3 INDUCTION OF PROTEASOME INHIBITOR SECRETION IN P. LUMINESCENS 61 6.3.1 TRIGGERING THE PATHOGENIC PHASE IN PHOTORHABDUS 61 6.3.2 ISOLATION AND STRUCTURE ELUCIDATION OF THE NATURAL PROTEASOME INHIBITORS 63 6.4 FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF CEPI AND GLBA 66 6.4.1 STRUCTURE ELUCIDATION BY 2D-NMR SPECTROSCOPY 66 6 TABLE OF CONTENTS 6.4.2 REVERSIBILITY ASSAYS, SELECTIVITY AND IC50 MEASUREMENTS 68 6.4.3 ANALYSIS OF THE YEAST 20S PROTEASOME : LIGAND COMPLEX STRUCTURES 70 6.4.4 TARGET SPECIFIC ADAPTATIONS OF SYRBACTINS 74 6.5 CELL VIABILITY AND PATHWAY SPECIFIC PROTEIN ACCUMULATION ASSAYS 79 6.5.1 CELL CYTOTOXICITY MEASUREMENTS 79 6.5.2 PATHWAY SELECTIVE PROTEIN ACCUMULATION ASSAY 81 7. DISCUSSION 83 7.1 DEVELOPMENT OF A TOOL FOR THE IDENTIFICATION OF CP INHIBITORS FROM NATURAL SOURCES 83 7.2 ISOLATION AND CHARACTERIZATION OF CEPI AND GLBA 86 8. REFERENCES 88 9 ABBREVIATIONS 95 10 PUBLICATIONS 97 11 ACKNOWLEDGEMENT 98 7
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spellingShingle	Stein, Martin Lorenz Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens
subject_GND	(DE-588)4113937-9
title	Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens
title_auth	Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens
title_exact_search	Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens
title_full	Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens Martin Lorenz Stein
title_fullStr	Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens Martin Lorenz Stein
title_full_unstemmed	Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens Martin Lorenz Stein
title_short	Detection, identification and characterization of the natural 20S proteasome inhibitors from Photorhabdus luminescens
title_sort	detection identification and characterization of the natural 20s proteasome inhibitors from photorhabdus luminescens
topic_facet	Hochschulschrift
url	http://mediatum.ub.tum.de/node?id=1177664 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20131216-1177664-0-6 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027003422&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT steinmartinlorenz detectionidentificationandcharacterizationofthenatural20sproteasomeinhibitorsfromphotorhabdusluminescens

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