Verfügbarkeit: Structural and functional characterization of the immunoproteasome

Structural and functional characterization of the immunoproteasome:

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Bibliographische Detailangaben
Beteilige Person:	Huber, Eva Maria (VerfasserIn)
Format:	Hochschulschrift/Dissertation Buch
Sprache:	Englisch
Veröffentlicht:	2013
Schlagwörter:	Autoimmunität Proteasen Hochschulschrift
Links:	http://mediatum.ub.tum.de/node?id=1130231 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130404-1130231-0-5 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025970275&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
Umfang:	101 S. Ill., graph. Darst.

Internformat

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Datensatz im Suchindex

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adam_text	IMAGE 1 TABLE OF CONTENTS TABLE OF CONTENTS 1 SUMMARY 8 2 ZUSAMMENFASSUNG 10 3 INTRODUCTION 12 3.1 INTRACELLULAR PROTEIN DEGRADATION AND ANTIGEN PRESENTATION 12 3.2 THE UBIQUITIN-PROTEASOME SYSTEM 13 3.2.1 POLYUBIQUITYLATION - A SIGNAL FOR DEGRADATION 13 3.2.2 SUBUNIT COMPOSITION AND ARCHITECTURE OF PROTEASOMES 14 3.2.3 PROTEOLYSIS BY 20S PROTEASOMES 16 3.2.4 IMMUNE FUNCTIONS OF 20S PROTEASOMES 18 3.3 20S PROTEASOMES: VALIDATED AND EMERGING DRUG TARGETS 19 3.4 TYPES OF PROTEASOME INHIBITORS 20 3.5 CLINICALLY RELEVANT PROTEASOME INHIBITORS 21 3.6 SUBUNIT-SPECIFIC PROTEASOME INHIBITORS AND THEIR THERAPEUTIC POTENTIAL 23 4 OBJECTIVE 25 5 MATERIALS AND METHODS 26 5.1 MATERIALS 26 5.1.1 CHEMICALS 26 5.1.2 ANTIBIOTICS 26 5.1.3 MEDIA 26 5.1.4 ENZYMES 27 5.1.5 PRIMER 28 5.1.6 STRAINS 29 5.1.7 PLASMIDS 29 5.1.8 FLUOROGENIC SUBSTRATES 30 5.1.9 DNA AND PROTEIN STANDARDS 31 5.1.10 INSTRUMENTS 31 5.1.11 COMPUTER SOFTWARE AND BIOINFORMATICS TOOLS 33 5.2 GENETIC METHODS 34 HTTP://D-NB.INFO/1038392969 IMAGE 2 TABLE OF CONTENTS 5.2.1 CULTIVATION AND LONG-TERM STORAGE OF ESCHERICHIA COLI 34 5.2.2 CULTIVATION AND LONG-TERM STORAGE OF SACCHAROMYCES CEREVISIAE 34 5.2.3 POLYMERASE CHAIN REACTION 34 5.2.4 SITE-DIRECTED MUTAGENESIS 36 5.2.5 AGAROSE GEL ELECTROPHORESIS 36 5.2.6 ISOLATION OF PLASMID DNA FROM E. COLI 37 5.2.7 PURIFICATION OF DNA 37 5.2.8 RESTRICTION DIGEST OF DNA 37 5.2.9 DETERMINATION OF DNA CONCENTRATION 38 5.2.10 LIGATION OF DNA FRAGMENTS 38 5.2.11 TRANSFORMATION OF E. COLI BY ELECTROPORATION 38 5.2.12 TRANSFORMATION OF S. CEREVISIAE 39 5.2.13 PLASMID SHUFFLING 39 5.2.14 DNA ISOLATION FROM S. CEREVISIAE 40 5.2.15 DNA SEQUENCING 40 5.3 PROTEIN CHEMISTRY AND ANALYTICS 41 5.3.1 PURIFICATION OF THE YEAST 20S PROTEASOME 41 5.3.2 PURIFICATION OF THE MURINE IMMUNO- AND CONSTITUTIVE 20S PROTEASOME 41 5.3.3 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS 42 5.3.4 DETERMINATION OF PROTEIN CONCENTRATION 43 5.4 PROTEASOME ACTIVITY TESTS 43 5.4.1 OVERLAY ASSAY 43 5.4.2 FLUORESCENCE ACTIVITY TEST AND DETERMINATION OF IC50 VALUES 44 5.5 PROTEIN CRYSTALLOGRAPHY 44 5.5.1 CRYSTALLIZATION OF THE YEAST 20S PROTEASOME 44 5.5.2 CRYSTALLIZATION OF THE MURINE 20S PROTEASOMES 45 5.5.3 DATA COLLECTION, PROCESSING AND STRUCTURE DETERMINATION 45 6 RESULTS 47 6.1 SEQUENCE ALIGNMENTS 47 6.2 X-RAY STRUCTURES OF THE MOUSE 20S IMMUNO- AND CONSTITUTIVE PROTEASOME 49 6.2.1 CRYSTALLIZATION AND STRUCTURE DETERMINATION 49 6.2.2 SUBUNIT ARCHITECTURE OF THE CCP AND ICP : 52 6.2.3 STRUCTURAL ANALYSIS OF THE SUBSTRATE BINDING CHANNELS 53 IMAGE 3 TABLE OF CONTENTS 6.3 INVESTIGATIONS ON THE P5I-SELECTIVE PROTEASOME INHIBITOR ONX 0914 57 6.3.1 INHIBITION OF 20S PROTEASOMES BY ONX 0914 57 6.3.2 PROTEASOME CORE PARTICLES IN COMPLEX WITH ONX 0914 57 6.3.3 MOLECULAR BASIS FOR THE SUBUNIT SELECTIVITY OF ONX 0914 59 6.3.4 STRUCTURAL ANALYSIS OF THE EPOXY KETONE REACTION MECHANISM 62 6.4 YEAST MUTAGENESIS STUDIES ON THE SUBUNITS 05C, \|35I AND P5T 66 6.4.1 MIMICKING THE 05C ACTIVE SITE 67 6.4.2 ANALYSIS OF THE P5I SUBSTRATE BINDING CHANNEL 69 6.4.3 PROBING SUBUNIT P5T 73 7 DISCUSSION 76 7.1 STRUCTURAL AND FUNCTIONAL DIFFERENCES BETWEEN THE THREE TYPES OF CPS 76 7.2 GUIDELINES FOR THE RATIONAL DESIGN OF CP-SELECTIVE INHIBITORS 78 8 BIBLIOGRAPHY 81 9 APPENDIX 90 10 ABBREVIATIONS ....96 11 PUBLICATIONS 99 12 ACKNOWLEDGEMENT 100
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spellingShingle	Huber, Eva Maria Structural and functional characterization of the immunoproteasome Autoimmunität (DE-588)4319435-7 gnd Proteasen (DE-588)4047521-9 gnd
subject_GND	(DE-588)4319435-7 (DE-588)4047521-9 (DE-588)4113937-9
title	Structural and functional characterization of the immunoproteasome
title_auth	Structural and functional characterization of the immunoproteasome
title_exact_search	Structural and functional characterization of the immunoproteasome
title_full	Structural and functional characterization of the immunoproteasome Eva Maria Huber
title_fullStr	Structural and functional characterization of the immunoproteasome Eva Maria Huber
title_full_unstemmed	Structural and functional characterization of the immunoproteasome Eva Maria Huber
title_short	Structural and functional characterization of the immunoproteasome
title_sort	structural and functional characterization of the immunoproteasome
topic	Autoimmunität (DE-588)4319435-7 gnd Proteasen (DE-588)4047521-9 gnd
topic_facet	Autoimmunität Proteasen Hochschulschrift
url	http://mediatum.ub.tum.de/node?id=1130231 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20130404-1130231-0-5 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025970275&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT huberevamaria structuralandfunctionalcharacterizationoftheimmunoproteasome

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