m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis:
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| Format: | Abschlussarbeit Mikrofilm Buch |
| Sprache: | English |
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Köln
2016
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| Beschreibung: | 2 Mikrofiches (135 Seiten) Illustrationen, Diagramme |
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Datensatz im Suchindex
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| adam_text | Titel: m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and
Autor: König, Tim
Jahr: 2016
Table of content
ABSTRACT.............................................................................................................................4
1 INTRODUCTION...................................................................................................................8
1.1 Mitochondrial protein biogenesis.................................................................................9
1.2 Proteases within mitochondria..................................................................................11
1.3 Quality control of mitochondrial function...................................................................13
1.4 Ion homeostasis in mitochondria - a critical role of Ca2+..........................................15
1.5 The molecular identity of the mitochondrial Ca2+ uniporter.......................................18
1.6 The m-AAA protease.................................................................................................20
1.6.1 From prokaryotes to humans..........................................................................20
1.6.2 Pathologies......................................................................................................22
1.6.3 Animal models and proposed cause of pathology..........................................23
1.7 Aim of the thesis........................................................................................................25
2 MATERIAL AND METHODS..............................................................................................26
2.1 Molecular biology......................................................................................................26
2.1.1 Cloning............................................................................................................26
2.1.2 RNA isolation and quantitative RT-PCFt..........................................................26
2.2 Biochemistry..............................................................................................................27
2.2.1 Preparation of protein extracts........................................................................27
2.2.2 Polyacrylamide-gel electrophoresis and immunoblotting................................27
2.2.3 Isolation of mitochondria.................................................................................28
2.2.4 Complexome profiling......................................................................................28
2.2.5 Solubilization of mitochondrial proteins...........................................................28
2.2.6 Oxygen consumption measurement...............................................................28
2.2.7 Purification of AFG3L2trap containing complexes.............................................29
2.2.8 Immunoprecipitation of MAIP1-MYC and MCU-FLAG....................................29
2.2.9 Determination of Ca2+ retention capacity........................................................29
2.2.10 Synthesis of35S-labeled proteins and MPP-cleavage...................................30
2.2.11 Mitochondrial protein import and chemical crosslinking................................30
2.2.12 Assembly ofMCU and EMRE.......................................................................30
2.2.12 Cell-free expression of MAIP1 and in vitro binding assays...........................31
2.3 Cell biology............................................31
2.3.1 Tissue culture..................................................................................................31
2.3.2 Transfection ofplasmids.................................................................................31
2.3.3 Transfection ofsiRNAs....................................................................................32
2.3.4 Generation of 2mtGCaMP6m expressing HeLa cells.....................................33
2.3.5 Immunocytochemistry.....................................................................................33
2.3.6 Transmission electron microscopy..................................................................33
2.3.7 Metabolic labeling of mitochondrial translation products.................................33
2.3.8 JC1 measurements.........................................................................................24
2.3.9 Ca2* and matrix pH measurements.................................................................34
2.3.10 Induction of apoptosis...................................................................................34
2.4 Mouse analysis.........................................................................................................34
2.4.1 Animal care and mouse handling....................................................................34
2.4.2 Mouse lines and breeding...............................................................................35
2.5 Bioinformatic analysis...............................................................................................35
2.6 Statistical analysis.....................................................................................................35
3 RESULTS...........................................................................................................................36
3.1 The membrane-associated matrix protein MAIP1 is an interactor rather
than a substrate of the m-AAA protease.........................................................................36
3.2 Loss of MAI PI or the m-AAA protease sensitizes cells towards apoptosis
independent of aberrant mitochondrial morphology........................................................44
3.3 MCU depletion restores apoptotic resistance of MAIP1- or m-AAA
protease-deficient cells...................................................................................................49
3.4 Accelerated mitochondrial Ca2+ overload during apoptosis induction in
MAIP1- or m-AAA protease-depleted HeLa cells............................................................51
3.5 Biogenesis and quality control of the MCU regulators MICU1 and MICU2...............56
3.6 Functionality of MCU depends on maturation of MICU1 and MICU2........................61
3.7 MAI PI assists sorting of EMRE in mitochondria and exerts chaperone-
like activity.......................................................................................................................64
3.8 The m-AAA protease limits MCU assembly by degrading the rate-limiting
subunit EMRE.................................................................................................... 70
3.9 Loss of the m-AAA protease sensitizes mitochondria towards MPTP
opening...........................................................................................................................77
3.10 Loss of the m-AAA protease alters MCU assembly and accelerates
MPTP opening in vivo.....................................................................................................82
4 DISCUSSION......................................................................................................................87
4.1 The m-AAA protease-MAIP1 complex assists in mitochondrial protein
biogenesis.......................................................................................................................88
4.2 Perturbed mitochondrial Ca2* homeostasis is the cause for
neurodegeneration in m-AAA protease-deficiency..........................................................91
4.3 Biogenesis and assembly of the mitochondrial Ca2* uniporter complex...................97
4.4 Implications of Ca2* disturbances as general cause of neurodegenerative
diseases........................................................................................................................102
REFERENCES.....................................................................................................................106
COMPLEXOME PROFILING...............................................................................................124
LIST OF ABBREVIATIONS.................................................................................................127
LIST OF FIGURES..............................................................................................................130
KURZZUSAMMENFASSUNG.............................................................................................132
ACKNOWLEDGMENT........................................................................................................133
EIDESSTATTLICHE ERKLARUNG....................................................................................134
CURRICULUM VITAE.........................................................................................................135
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| dewey-ones | 571 - Physiology & related subjects 572 - Biochemistry |
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| discipline | Biologie |
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| spelling | König, Tim 1986- Verfasser (DE-588)1111818452 aut m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis vorgelegt von Tim König Köln 2016 2 Mikrofiches (135 Seiten) Illustrationen, Diagramme txt rdacontent h rdamedia he rdacarrier 24x Dissertation Universität zu Köln 2016 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/112114053X/04 Inhaltsverzeichnis HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029669386&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | König, Tim 1986- m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis |
| subject_GND | (DE-588)4113937-9 |
| title | m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis |
| title_auth | m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis |
| title_exact_search | m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis |
| title_full | m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis vorgelegt von Tim König |
| title_fullStr | m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis vorgelegt von Tim König |
| title_full_unstemmed | m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis vorgelegt von Tim König |
| title_short | m-AAA proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and Ca2+ homeostasis |
| title_sort | m aaa proteases protect against neurodegeneration by regulating mitochondrial protein biogenesis and ca2 homeostasis |
| topic_facet | Hochschulschrift |
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