Molecular characterization of petide GPCRs: structure-activity relations relationships and Subtype selectivity of the neuropetide Y system
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| Format: | Thesis Electronic Software eBook |
| Language: | English |
| Published: |
Leipzig
[2014]
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| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | 1 CD-ROM (190 Seiten) Illustrationen |
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| 100 | 1 | |a Pedragosa Badia, Xavier |d 1985- |e Verfasser |0 (DE-588)1117103781 |4 aut | |
| 245 | 1 | 0 | |a Molecular characterization of petide GPCRs |b structure-activity relations relationships and Subtype selectivity of the neuropetide Y system |c vorgelgt von Master in Molecular Biotechnology Xavier Pedragosa Badia |
| 264 | 1 | |a Leipzig |c [2014] | |
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| 502 | |b Dissertation |c Universität Leipzig |d 2014 | ||
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| adam_text | TABLE OF CONTENT
ABBREVIATIONS...............................................................................................................................9
BIBLIOGRAPHISCHE BESCHREIBUNG
.................................................................................
13
SUMMARY.........................................................................................................................................
14
ZUSAMMENFASSUNG...................................................................................................................19
AIM OF THE
THESIS......................................................................................................................25
1. CHAPTER
1................................................................................................................................26
1.1
SUMMARY......................................................................................................................
27
1.2 INTRODUCTION TO THE NEUROPEPTIDE Y
FAMILY......................................................................28
1.3 HOW TO OBTAIN SELECTIVE LIGANDS FOR NPY
RECEPTORS......................................................32
1.3.1 GENERAL
STRATEGIES.......................................................................................................32
1.3.2 YI
RECEPTOR...................................................................................................................33
1.3.3 Y2
RECEPTOR...................................................................................................................36
1.3.4 Y4
RECEPTOR...................................................................................................................37
1.3.5 Y5
RECEPTOR...................................................................................................................38
1.4 HOW TO IDENTIFY RELEVANT RESIDUES ON THE RECEPTOR FOR BINDING AND
SUBTYPE
SELECTIVITY..........................................................................................................................................40
1.4.1 GENERAL
STRATEGIES......................................................................................................
40
1.4.2 Y
1
RECEPTOR...................................................................................................................41
1.4.3 Y2
RECEPTOR...................................................................................................................42
1.4.4 Y4
RECEPTOR...................................................................................................................44
1.4.5 Y5
RECEPTOR...................................................................................................................45
1.5 CONCLUSIONS AND
PERSPECTIVES............................................................................................46
1.6
ACKNOWLEDGMENT.................................................................................................................47
1.7
REFERENCES............................................................................................................................48
2. CHAPTER 2
................................................................................................................................63
2.1
SUMMARY.................................................................................................................................64
2.2 INTRODUCTION,
65
2.3 EXPERIMENTAL
PROCEDURES...................................................................................................67
2.3.1 PEPTIDE
SYNTHESIS...................................................................................................67
2.3.2 PREPARATION OF I1Y4R MUTANTS
..............................................................................
67
2.3.3 CELL
CULTURE..............................................................................................................68
2.3.4 FLUORESCENCE MICROSCOPY
STUDIES.......................................................................68
2.3.5 SIGNAL TRANSDUCTION
ASSAYS...................................................................................68
2.3.6 FOURTEEN EXPERIMENTAL GPCR STRUCTURES WERE CONSIDERED AS TEMPLATES
FOR
I1Y4R COMPARATIVE
MODELING....................................................................................................68
2.3.7 MISSING ATOM COORDINATES WERE CONSTRUCTED USING ROSETTA LOOP
CONSTRUCTION
PROTOCOLS.......................................................................................................................................69
2.3.8 DOCKING OF PANCREATIC POLYPEPTIDE (PP) INTO THE COMPARATIVE MODEL
OF
HY4R...............................................................................................................................................69
2.3.9 C-TERMINAL RESIDUES OF HPP WERE ADDED USING DE NOVO FOLDING WITH
EXPERIMENTAL
RESTRAINTS...............................................................................................................70
2.3.10 MODELS WERE RELAXED USING ATOMIC-RESOLUTION EXPERIMENTAL
RESTRAINTS
......
70
2.4
RESULTS....................................................................................................................................73
2.4.1 TYR2 64 OF TM2 INTERACTS WITH
TYR27......................................................................73
2.4.2 OTHER POSITIONS HIGHLIGHT THE IMPORTANCE OF
ECLI.........................................74
2.4.3 THE TESTED RESIDUES IN TM3, ECL2 AND TM5 DO NOT PLAY A RELEVANT
ROLE IN THE
BINDING
POCKET.............................................................................................................................77
2.4.4 TM
OE
PLAYS A CRUCIAL ROLE IN BUILDING THE BINDING POCKET
..............................
79
2.4.5
TM7
IS A CONTACT POINT OF HPP IN THE HY4R, ASN7 32 INTERACTS WITH ARG33 OF
HPP.................................................................................................................................................79
2.4.6 PHE7 35 INTERACTS WITH ARG33 AS WELL AS TYR36
....................................................
82
2.4.7 DOCKING OF PP TO THE HY4R COMPARATIVE MODEL
.............................................
85
2.5
DISCUSSION.............................................................................................................................90
2.6
ACKNOWLEDGEMENTS.............................................................................................................94
2.7 REFERENCES,
95
2.8 SUPPLEMENTARY
INFORMATION............................................................................................102
2.8.1 EXPERIMENTAL PROCEDURES
........................................................................................
102
2.9
APPENDIX...........................................................................................................................
111
2.9.1
INTRODUCTION...............................................................................................................111
2.9.2 FURTHER AMINO ACID EXCHANGES IN
ECL2...............................................................ILL
2.9.3 DOUBLE MUTAGENESIS IN POSITIONS ASP6 59 AND ASN7 32
...................................... 1 12
2.9.4 FURTHER INVESTIGATIONS AT POSITION 35 OF
HPP......................................................113
2.9.5
CONCLUSIONS..............................................................................................................113
3. CHAPTER 3
.....................................................................................
.
......................................
114
3.1
SUMMARY.............................................................................................................................115
3.2
INTRODUCTION........................................................................................................................
116
3.3 EXPERIMENTAL
PROCEDURES................................................................................................118
3.3.1 PEPTIDE
SYNTHESIS......................................................................................................118
3.3.2 GENERATION OF MUTANT AND CHIMERIC Y-RECEPTORS
..............................................
118
3.3.3 CELL
CULTURE................................................................................................................119
3.3.4 FLUORESCENCE MICROSCOPY STUDIES
.........................................................................
119
3.3.5 SIGNAL TRANSDUCTION
ASSAYS....................................................................................120
3.3.6 MEMBRANE PREPARATIONS FOR RADIOLIGAND BINDING
ASSAYS...................................120
3.3.7 RADIOLIGAND BINDING
ASSAYS....................................................................................121
3.4
RESULTS.................................................................................................................................
122
3.4.1 DESIGN OF CHIMERIC RECEPTORS
.................................................................................
122
3.4.2
ECL3
DISCRIMINATES PNPY BINDING IN THE Y4R SUBTYPE
..................................
123
3.4.3 CRITICAL AMINO ACIDS IN THE ECL3 OF HYR SUBTYPES
.........................................
124
3.4.4 HYIR/HY4R SEGMENT CHIMERA REVEAL AN N-TERMINAL FRAGMENT THAT
PREVENTS
PNPY ACTIVATION ON HY4R
.....................................................................................................124
3.4.5 COMBINATION OF NT TO ECE, AND ECL3 FRAGMENTS OF HY,R IN HY4R
CHIMERA
RESULTED IN EQUAL ACTIVITY FOR BOTH, PNPY AND
HPP............................................................125
3.4.6 THREE AMINO ACIDS OF THE TM2 OF Y4R PREVENT HIGH POTENCY OF PNPY
.......
126
3.4.7 RADIOLIGAND BINDING ASSAYS REVEAL THAT HY4R RECEPTOR CONSTRUCTS
WITH HYIR
LIKE MODIFICATIONS ON TM2 BIND 125I-PPYY WITH MORE AFFINITY COMPARED TO
HY4R
WT 128
3.5
DISCUSSION...........................................................................................................................132
3.6
ACKNOWLEDGEMENTS...........................................................................................................136
3.7
REFERENCES...........................................................................................................................137
4. CHAPTER 4
...............................................................................................................................142
4.1
SUMMARY.............................................................................................................................
143
4.2
INTRODUCTION.........................................................................................................................144
4.3 EXPERIMENTAL
PROCEDURES.................................................................................................149
4.3.1 PEPTIDE
SYNTHESIS.......................................................................................................149
4.3.2 GENERATION OF PRRP RECEPTOR
MUTANTS...................................................................149
4.3.3 CELL
CULTURE.................................................................................................................149
4.3.4 REACTION WITH MTSEA, MTSES AND
MTSET...................................................150
4.3.5 RESAZURIN-BASED CELL VIABILITY ASSAY
.....................................................................
150
4.3.6 SIGNAL TRANSDUCTION
ASSAY........................................................................................151
4.3.7 FLUORESCENCE
MICROSCOPY........................................................................................151
4.4
RESULTS..................................................................................................................................152
4.4.1 VERIFICATION OF EIGHT CRUCIAL RESIDUES FOR RECEPTOR ACTIVITY
AND/OR LIGAND
BINDING.........................................................................................................................................152
4.4.2 ESTABLISHING THE SET UP FOR THE SUBSTITUTED-CYSTEINE ACCESSIBILITY
METHOD
(SCAM) COUPLED TO AN IP ACCUMULATION
ASSAY...................................................................154
4.4.3 EFFECT OF CYSTEINE-SUBSTITUTIONS ON CRUCIAL RECEPTOR RESIDUES IN
TM 1, 5, 6, 7
AND
ECL2....................................................................................................................................155
4.4.4 EFFECT OF MTSEA, MTSES AND MTSET TREATMENT ON W2JIC, W5 28C, E5
26C
AND F6
54C....................................................................................................................................157
4.4.5 MTS-REAGENTS ARE CAPABLE TO MODIFY THE BINDING POCKET AND RECEPTOR
ACTIVATION AT D6 59C AND Y5 38C MUTANTS OF PRRPR
............................................................
157
4.4.6 Q735 OF PRRPR MODULATES RECEPTOR POTENCY AND EFFICACY IN A SIZE
AND NOT
CHARGE DEPENDENT
MANNER.......................................................................................................160
4.5
DISCUSSION...........................................................................................................................162
4.6
ACKNOWLEDGEMENT............................................................................................................167
4.7
REFERENCES...........................................................................................................................168
4.8 SUPPLEMENTARY
INFORMATION............................................................................................174
4.8.1
RESULTS........................................................................................................................
174
PUBLICATIONS AND
PRESENTATIONS..............................................................................175
CURRICULUM
VITAE..................................................................................................................178
SELBSTSTAENDIGKEITSERKLAERUNG
.................................................................................
179
ACKNOWLEDGEMENT...............................................................................................................180
|
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| author | Pedragosa Badia, Xavier 1985- |
| author_GND | (DE-588)1117103781 |
| author_facet | Pedragosa Badia, Xavier 1985- |
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| author_sort | Pedragosa Badia, Xavier 1985- |
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| discipline | Biologie |
| format | Thesis Electronic Software eBook |
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| spelling | Pedragosa Badia, Xavier 1985- Verfasser (DE-588)1117103781 aut Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system vorgelgt von Master in Molecular Biotechnology Xavier Pedragosa Badia Leipzig [2014] 1 CD-ROM (190 Seiten) Illustrationen txt rdacontent c rdamedia cd rdacarrier Dissertation Universität Leipzig 2014 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1117104206/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030073457&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Pedragosa Badia, Xavier 1985- Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system |
| subject_GND | (DE-588)4113937-9 |
| title | Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system |
| title_auth | Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system |
| title_exact_search | Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system |
| title_full | Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system vorgelgt von Master in Molecular Biotechnology Xavier Pedragosa Badia |
| title_fullStr | Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system vorgelgt von Master in Molecular Biotechnology Xavier Pedragosa Badia |
| title_full_unstemmed | Molecular characterization of petide GPCRs structure-activity relations relationships and Subtype selectivity of the neuropetide Y system vorgelgt von Master in Molecular Biotechnology Xavier Pedragosa Badia |
| title_short | Molecular characterization of petide GPCRs |
| title_sort | molecular characterization of petide gpcrs structure activity relations relationships and subtype selectivity of the neuropetide y system |
| title_sub | structure-activity relations relationships and Subtype selectivity of the neuropetide Y system |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1117104206/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030073457&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT pedragosabadiaxavier molecularcharacterizationofpetidegpcrsstructureactivityrelationsrelationshipsandsubtypeselectivityoftheneuropetideysystem |
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