Verfügbarkeit: The role of translational velocity in mutant protein folding

The role of translational velocity in mutant protein folding: how CFTR translation modulates its folding

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1. Verfasser:	Rauscher, Robert (VerfasserIn)
Format:	Abschlussarbeit Buch
Sprache:	English
Veröffentlicht:	Hamburg [2018]
Schlagworte:	Hochschulschrift
Online-Zugang:	Inhaltsverzeichnis Inhaltsverzeichnis
Beschreibung:	96, XVII Seiten Illustrationen, Diagramme 21 cm

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adam_text	TABLE OF CONTENTS PUBLICATIONS................................................................................................. II TABLE OF CONTENTS....................................................................................ILL LIST OF FIGURES........................................................................................... VI LIST OF TABLES........................................................................................... VIII LIST OF ABBREVIATIONS..............................................................................IX ZUSAMMENFASSUNG ........................................................ XI ABSTRACT......................................................................................................XII 1. INTRODUCTION..........................................................................................1 1.1. TRANSLATION........................................................................................................................ 1 1.1.1. THE PHASES OF EUKARYOTIC TRANSLATION....................................................................... 1 1.1.2. FEATURES THAT MODULATE TRANSLATIONAL EFFICIENCY AND LOCAL VELOCITY ........................ 3 1.1.3. COTRANSLATIONAL PROTEIN FOLDING.................................................................................. 6 1.2. THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR ............................... 8 1.2.1. CFTR BIOGENESIS AND FOLDING ................................................................................... 8 1.2.2. CFTR FUNCTION..........................................................................................................10 1.2.3. MUTATIONS IN C FTR ................................................................................................... 11 1.2.4. CYSTIC FIBROSIS AND CFTR RELATED DISORDERS ........................................................ 13 1.2.5. DEGRADATION OF MISFOLDED CFTR .............................................................................. 14 1.2.6. PHARMACOLOGICAL TREATMENT OF CYSTIC FIBROSIS ....................................................... 16 1.3. ER-STRESS AND THE UNFOLDED PROTEIN RESPONSE .................................. 17 1.4. AIM OF THIS THESIS...........................................................................................................20 2. RESULTS....................................... . ...................................................... - 21 2.1. SEQUENCE FEATURES INFLUENCE TRANSLATIONAL VELOCITY.................................................21 2.1.1. CFTR TRANSLATION VELOCITY IS NOT UNIFORM ................................................................21 2.1.2. MEASURES OF TRANSLATIONAL VELOCITY BARELY CORRELATE WITH ONE ANOTHER .................. 22 2.2. TOWARDS DEEPER COVERAGE OF SPECIFIC TRANSCRIPTS IN RIBOSEQ EXPERIMENTS ...... 25 2.3. TRANSLATIONAL VELOCITY IS EMBEDDED INTO THE CFTR TRANSCRIPT .............................. 28 2.4. EFFECTS OF ALTERED TRANSLATIONAL VELOCITY ALONG THE CFTR TRANSCRIPT .................... 30 2.4.1. SSNPS OCCUR AT DIFFERENT FREQUENCIES.....................................................................30 2.4.2. MUTATIONS DIFFERENTIALLY AFFECT CFTR STEADY-STATE EXPRESSION AND MATURATION... 32 2.4.3. THE SSNP T2562G DIFFERENTIALLY AFFECTS WILD-TYPE AND MUTANT CFTR VARIANTS .33 2.4.4. THE T2562G SSNP AMELIORATES CFTR ACTIVITY OF SPECIFIC CFTR VARIANTS ......... 36 2.4.5. THE T2562G SSNP AUGMENTS STABILITY AND MUTANT CFTR VARIANTS ..................... 38 2.4.6. THE T2562G SSNP ENHANCED THE EFFECT OF CORRECTOR ON CFTR VARIANTS WITH CF- CAUSING M UTATION.......................................................................................................................40 2.5. MECHANISTIC INSIGHTS INTO RPL12 KNOCK-DOWN MEDIATED CFTR RESCUE .............. 42 2.6. CFTR MISFOLDING INDUCES TRANSCRIPTOME CHANGES..................................................46 3. DISCUSSION..............................................................................................52 3.1. TRANSLATIONAL DYNAMICS IN CFTR FOLDING....................................................................52 3.1.1. CFTR BIOSYNTHESIS IS NON-UNIFORM.....................................................................52 3.1.2. IMPERFECT CORRELATION OF VARIOUS MEASURES OF TRANSLATION VELOCITY ............... 53 3.1.3. TERIBO-SEQ PROVIDES INFORMATION ON RIBOSOME TRAFFIC FOR INDIVIDUAL TRANSCRIPTS................................................................................................................................ 54 3.1.4. TRANSLATIONAL SPEED IS EMBEDDED IN THE CFTR TRANSCRIPT...............................55 3.2. SSNPS FREQUENCIES IN PATIENTS DEVIATE FROM THESE IN HEALTHY POPULATION .......... 56 3.3. THE T2562G SSNP DIFFERENTIALLY AFFECTS DIFFERENT CFTR VARIANTS WITH A MAJOR CF MUTATION................................................................................................................ 57 3.4. RPL12-MEDIATED CODON-SPECIFIC TRANSLATIONAL SLOW-DOWN RESHAPES THE CFTR TRANSLATIONAL LANDSCAPE.............................................................................................................. 59 3.5. CFTR MISFOLDING COMPROMISES THE STRESS RESPONSE ............................................. 60 4. MATERIAL AND METHODS .................................................................... 63 4.1. MATERIALS............................................................................................................................63 4.1.1. REAGENTS AND CHEMICALS.........................................................................................63 4.1.2. ENZYMES AND BUFFERS............................................................................................... 63 4.1.3. ANTIBODIES ................................................. 64 4.1.4. KITS............................................................................................................................. 64 4.1.5. OLIGONUCLEOTIDES ................................................................... 65 4.1.6. BUFFERS AND REAGENTS 69 4.2. METHODS.................................................... 72 4.2.1. CELL CULTURE............................................................................................................... 72 4.2.2. RIBOSOME PROFILING AND MRNA SEQUENCING .......................................................... 72 4.3. BIOCHEMICAL EXPERIMENTS ............................................................................................. 75 4.3.1. CLONING AND TRANSFECTION......................................................................................... 75 4.3.2. WESTERN BLOTTING.......................................................................................................76 4.3.3. ISOLATION OF GENOMIC DNA......................................... 76 4.3.4. SPLICING ASSAY.......................................................................................................... 77 4.3.5. THERMAL AGGREGATION PROPENSITY ASSAY..................................................................77 4.3.6. PARTIAL PERMEABILIZATION OF THE CELLS AND LIMITED PROTEOLYSIS ................................. 77 4.3.7. ACTIVITY MEASUREMENTS............................................................................................ 78 4.3.6. SHORT-CIRCUIT CURRENT (LSC) MEASUREMENT...................................................................78 4.3.9. TOTAL RNA EXTRACTION, CDNA SYNTHESIS AND QUANTITATIVE PC R .............................. 78 4.3.10. TRNA TAILORED MICROARRAYS........................................................................................79 LIST OF REFERENCES ............... 80 SUPPLEMENTARY INFORMATION...............................................................92 LIST OF HAZARDOUS SUBSTANCES USED IN THE STUDY .................. XII ACKNOWLEDGEMENTS..............................................................................XVI DECLARATION ON OATH .......................................................................... XVII
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spelling	Rauscher, Robert Verfasser (DE-588)1147688109 aut The role of translational velocity in mutant protein folding how CFTR translation modulates its folding vorgelegt von Robert Rauscher, M.Sc. Hamburg [2018] 96, XVII Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Hamburg 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1163625795/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030825887&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Rauscher, Robert The role of translational velocity in mutant protein folding how CFTR translation modulates its folding
subject_GND	(DE-588)4113937-9
title	The role of translational velocity in mutant protein folding how CFTR translation modulates its folding
title_auth	The role of translational velocity in mutant protein folding how CFTR translation modulates its folding
title_exact_search	The role of translational velocity in mutant protein folding how CFTR translation modulates its folding
title_full	The role of translational velocity in mutant protein folding how CFTR translation modulates its folding vorgelegt von Robert Rauscher, M.Sc.
title_fullStr	The role of translational velocity in mutant protein folding how CFTR translation modulates its folding vorgelegt von Robert Rauscher, M.Sc.
title_full_unstemmed	The role of translational velocity in mutant protein folding how CFTR translation modulates its folding vorgelegt von Robert Rauscher, M.Sc.
title_short	The role of translational velocity in mutant protein folding
title_sort	the role of translational velocity in mutant protein folding how cftr translation modulates its folding
title_sub	how CFTR translation modulates its folding
topic_facet	Hochschulschrift
url	http://d-nb.info/1163625795/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030825887&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
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