Roles of Munc18-1 in synaptic vesicle priming and disease:
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| 035 | |a (OCoLC)1372152447 | ||
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| 100 | 1 | |a André, Timon |d 1989- |e Verfasser |0 (DE-588)1160962685 |4 aut | |
| 245 | 1 | 0 | |a Roles of Munc18-1 in synaptic vesicle priming and disease |c presented by Timon Christian André (M.Sc.) |
| 264 | 1 | |a Heidelberg |c 2022 | |
| 300 | |a iii, 107 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruprecht-Karls-University Heidelberg |d 2022 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1279866772/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034909706&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-034909706 | ||
| 883 | 2 | |8 1\p |a dnb |d 20230215 |q DE-101 |u https://d-nb.info/provenance/plan#dnb | |
| 883 | 2 | |8 2\p |a dnb |d 20230215 |q DE-101 |u https://d-nb.info/provenance/plan#dnb | |
Datensatz im Suchindex
| _version_ | 1804186417782325248 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
1
SUMMARY
........................................................................................................................................
1
2
ZUSAMMENFASSUNG
....................................................................................................................
2
3
INTRODUCTION
................................................................................................................................
3
3.1
SYNAPTIC
TRANSMISSION
...............................................
3
3.1.1
SYNAPTIC
VESICLES
AND
THE
ACTIVE
ZONE
....................................................................................................
3
3.1.2
SYNAPTIC
VESICLE
FUSION
............................................................................................................................
6
3.1.2.1
SNARE
PROTEINS
-
THE
CORE
FUSION
MACHINERY
...............................................................................
6
3.1.2.2
VESICLE
DOCKING
...............................................................................................................................
8
3.1.2.3
PRIMING
............................................................................................................................................
9
3.1.2.3.1
MUNCL8-L
TEMPLATES
SNARE
COMPLEX
ASSEMBLY
..................................................................
11
3.1.2.3.2
THE
MUNCL8-L-SYNTAXIN-L
N-PEPTIDE
INTERACTION
.................................................................
13
3.1.2.3.3
MUND3-1
................................................................................................................................14
3.1.2.4
LATE
PRIMING
STEPS
........................................................................................................................
14
3.1.2.5
SNARE
COMPLEX
DISASSEMBLY
.......................................................................................................
15
3.2
MUNCL8-L
PHOSPHORYLATION
.........................................................................................................
16
3.3
MUNCL8-L
AND
DISEASE
..................................................................................................................
16
3.3.1
STXBP1
SYNDROME
..........................
17
3.3.2
PREDICTING
PROTEIN
STABILITY
....................................................................................................................
18
3.4
PROJECT
AIMS
.....................................................................................................................................
18
4
MATERIAL
AND
METHODS
........................................................................................................
20
4.1
PROTEIN
EXPRESSION
AND
LYSIS
.........................................................................................................
20
4.2
PROTEIN
PURIFICATION
........................................................................................................................
21
4.2.1
MUNCL8-L
...............................................................................................................................................
22
4.2.2
MUNCL8-L
BPA
MUTANTS
.........................................................................................................................
22
4.2.3
SNAP-25
..................................................................................................................................................
24
4.2.4
SYNTAXIN-1
...............................................................................................................................
25
4.2.5
T-SNARE
...................................................................................................................................................
25
4.2.6
VAMP2/VAMP8
.....................................................................................................................................
27
4.2.7
SYNAPTOTAGMIN-1
...................................................................................................................................
28
4.2.8
RAB3A
.......................................................................................................................................................
29
4.3
SDS-PAGE
(SODIUM
DODECYL
SULFATE-POLYACRYLAMIDE
GEL
ELECTROPHORESIS)
............................
30
4.3.1
DETERMINATION
OF
PROTEIN
CONCENTRATIONS
...........................................................................................
31
4.4
LIPID
MODIFICATION
OF
RAB3A
..........................................................................................................
31
4.5
PROTEIN
STABILITY
MEASUREMENTS
USING
DSF
.................................................................................
31
4.6
GEL
FILTRATION
...................................................................................................................................
32
4.7
PREPARATION
OF
PROTEOLIPOSOMES
..................................................................................................
33
4.7.1
V-SNARE
SUVS
........................................................................................................................................
34
4.7.2
T-SNARE/SYNTAXIN-1
SUVS
(FOR
ELECTRO-SWELLING):
................................................................................
35
4.7.3
DETERMINATION
OF
PROTEIN
:
IIPID
RATIOS
OF
THE
SUVS
.............................................................................
36
4.8
ELECTRO-SWELLING
OF
T-SNARE/SYNTAXIN-1
GUVS
..........................................................................
36
4.9
SUV/GUV
FUSION
ASSAY
...................................................................................................................
37
4.10
SUV/GUV
DOCKING
ASSAY
.................................................................................................................
38
4.10.1
MUNCL8-L
DOCKING
ASSAY
..................................................................................................................
38
4.10.2
RAB3A/RIM1
DOCKING
ASSAY
..............................................................................................................
39
4.11
CROSSLINKING
OF
MUNCL8-L
BPA
MUTANTS
......................................................................................
39
4.12
WESTERN
BLOT
...................................................................................................................................
40
4.13
STATISTICAL
ANALYSIS
...........................................................................................................................
41
4.14
CONTRIBUTIONS
BY
OTHER
RESEARCHERS
.............................................................................................
41
5
RESULTS
..........................................................................................................................................
42
5.1
RECONSTITUTED
LIPOSOME
FUSION
ASSAY
..........................................................................................
42
5.2
STAGE-SPECIFIC
EFFECTS
OF
MUNCL8-L
DEPEND
ON
THE
RECOGNITION
OF
THE
COGNATE
V-SNARE
43
5.3
MOLECULAR
DETAILS
OF
THE
VAMP2*MUNCL8-L
INTERACTION
...........................................................
45
5.3.1
SITE-SPECIFIC
PHOTO-CROSSLINKING
MAPS
THE
VAMP2-MUNCL8-1
BINDING
INTERFACE
IN
THE
RECONSTITUTED
TEMPLATE
COMPLEX
........................................................................................................................
45
5.3.2
Q301
IN
HELIX
11
OF
MUNCL8-L
IS
ESSENTIAL
FOR
VAMP2
BINDING
.........................................................
48
5.3.3
THE
VAMP2-MUNCL8-1
INTERACTION
IS
REQUIRED
TO
PROMOTE
LIPID-MIXING
IN
RECONSTITUTED
MEMBRANE
FUSION
ASSAYS
......................................................................................................................................
50
5.4
MUNCL8-L
PHOSPHORYLATION
..........................................................................................................
52
5.4.1
A
PHOSPHOMIMETIC
MUTATION
IN
HELIX
12
OF
MUNCL8-L
REGULATES
THE
TEMPLATING
FUNCTION
IN
A
STAGE-SPECIFIC
MANNER
..........................................................................................................................................
52
5.4.2
MUNCL8-L
S345BPA
SHOWS
AN
INTRAMOLECULAR
CROSS-LINK
TO
THE
FURLED
LOOP
....................................
53
5.4.3
CONSTITUTIVELY
OPEN
MUND8-1
AND
SYNTAXIN-1
MUTANTS
COUNTERACT
THE
INHIBITORY
EFFECT
OF
THE
S345E
MUTATION
.........................................................................................................................
57
5.5
STXBP1
SYNDROME
..........................................................................................................................
60
5.5.1
MISSENSE
MUTATIONS
IN
MUNCL8-CAUSE
STXBP1
SYNDROME
.................................................................
61
5.5.2
ANALYSING
THE
EFFECTS
OF
MUNL8-1
MUTATIONS
ON
PROTEIN
STABILITY
.....................................................
61
5.5.3
THE
ANALYZED
MUNCL8-L
MUTANTS
DO
NOT
AGGREGATE
IN
VITRO
...............................................................
62
5.5.4
DIFFERENTIAL
EFFECTS
OF
MUNCL8-L
MUTATIONS
ON
SNARE
FUSION
............................................................64
5.5.5
DISEASE-CAUSING
MUNCL8-L
MUTATIONS
CAUSE
STAGE-SPECIFIC
EFFECTS
..................................................
66
5.5.6
PREDICTING
MUNCL8-L
MUTATION
SEVERITY
...............................................................................................
67
5.6
RECONSTITUTING
VESICLE
TETHERING
...........................................................................................
69
5.6.1
RAB3A
NUCLEOTIDE
EXCHANGE
AND
LIPID
MODIFICATION
.............................................................................
69
5.6.2
RAB3A
AND
RIM1
PROMOTE
SUV-GUV
ASSOCIATION
IN
A
NUCLEOTIDE-SPECIFIC
MANNER
..........................
70
5.6.3
THE
RAB3A-RIM1
INTERACTION
STIMULATES
SNARE-MEDIATED
FUSION
.....................................................
71
II
6
DISCUSSION
...................................................................................................................................
74
6.1
MUNCL8-1*VAMP2
INTERACTIONS
ARE
ESSENTIAL
FOR
TEMPLATE
COMPLEX
FORMATION
...............
74
6.2
PHOSPHORYLATION
OF
MUNC18-1
NEGATIVELY
INFLUENCES
THE
DYNAMICS
OF
HELIX12
AND
BLOCKS
FUSION
IN
A
STAGE-SPECIFIC
MANNER
..............................................................................................................
76
6.3
WHEN
DOES
SNAP-25
ENTER
THE
STAGE?
........................................................................................
78
6.4
MISSENSE
MUTATIONS
IN
MUNCL8-L
CAUSE
STXBPL-SYNDROME
...................................................
80
6.4.1
DISEASE-CAUSING
MUNCL8-L
MUTATIONS
LEAD
TO
PROTEIN
INSTABILITY
.....................................................
80
6.4.2
THE
TESTED
DISEASE-CAUSING
MUTATIONS
DO
NOT
SHOW
DOMINANT
NEGATIVE
PROPERTIES
.......................
81
6.4.3
DISEASE-CAUSING
MUNCL8-L
MUTANTS
SHOW
IMPAIRED
STIMULATION
ON
SNARE-MEDIATED
FUSION
.......
81
6.4.4
ALTERNATIVE
DISEASE
MECHANISMS
FOR
MUNCL8-L
T361I,
H445P,
AND
L446F.......................................
82
6.4.5
IN
SILICO
MUTANT
PREDICTION
TO
CLASSIFY
STXBP1
MUTATIONS
...................................................................
84
6.5
RECONSTITUTING
SYNAPTIC
VESICLE
TETHERING
..................................................................................
84
7
APPENDIX
.......................................................................................................................................
87
7.1
LIST
OF
FIGURES
....................................................................................................................................
87
7.2
LIST
OF
TABLES
.....................................................................................................................................
88
7.3
ABBREVIATIONS
..................................................................................................................................
88
7.4
LITERATURE
..........................................................................................................................................
90
8
PUBLICATIONS
.............................................................................................................................
106
9
ACKNOWLEDGEMENTS.
.............................................................................................................
107
III
|
| adam_txt |
TABLE
OF
CONTENTS
1
SUMMARY
.
1
2
ZUSAMMENFASSUNG
.
2
3
INTRODUCTION
.
3
3.1
SYNAPTIC
TRANSMISSION
.
3
3.1.1
SYNAPTIC
VESICLES
AND
THE
ACTIVE
ZONE
.
3
3.1.2
SYNAPTIC
VESICLE
FUSION
.
6
3.1.2.1
SNARE
PROTEINS
-
THE
CORE
FUSION
MACHINERY
.
6
3.1.2.2
VESICLE
DOCKING
.
8
3.1.2.3
PRIMING
.
9
3.1.2.3.1
MUNCL8-L
TEMPLATES
SNARE
COMPLEX
ASSEMBLY
.
11
3.1.2.3.2
THE
MUNCL8-L-SYNTAXIN-L
N-PEPTIDE
INTERACTION
.
13
3.1.2.3.3
MUND3-1
.14
3.1.2.4
LATE
PRIMING
STEPS
.
14
3.1.2.5
SNARE
COMPLEX
DISASSEMBLY
.
15
3.2
MUNCL8-L
PHOSPHORYLATION
.
16
3.3
MUNCL8-L
AND
DISEASE
.
16
3.3.1
STXBP1
SYNDROME
.
17
3.3.2
PREDICTING
PROTEIN
STABILITY
.
18
3.4
PROJECT
AIMS
.
18
4
MATERIAL
AND
METHODS
.
20
4.1
PROTEIN
EXPRESSION
AND
LYSIS
.
20
4.2
PROTEIN
PURIFICATION
.
21
4.2.1
MUNCL8-L
.
22
4.2.2
MUNCL8-L
BPA
MUTANTS
.
22
4.2.3
SNAP-25
.
24
4.2.4
SYNTAXIN-1
.
25
4.2.5
T-SNARE
.
25
4.2.6
VAMP2/VAMP8
.
27
4.2.7
SYNAPTOTAGMIN-1
.
28
4.2.8
RAB3A
.
29
4.3
SDS-PAGE
(SODIUM
DODECYL
SULFATE-POLYACRYLAMIDE
GEL
ELECTROPHORESIS)
.
30
4.3.1
DETERMINATION
OF
PROTEIN
CONCENTRATIONS
.
31
4.4
LIPID
MODIFICATION
OF
RAB3A
.
31
4.5
PROTEIN
STABILITY
MEASUREMENTS
USING
DSF
.
31
4.6
GEL
FILTRATION
.
32
4.7
PREPARATION
OF
PROTEOLIPOSOMES
.
33
4.7.1
V-SNARE
SUVS
.
34
4.7.2
T-SNARE/SYNTAXIN-1
SUVS
(FOR
ELECTRO-SWELLING):
.
35
4.7.3
DETERMINATION
OF
PROTEIN
:
IIPID
RATIOS
OF
THE
SUVS
.
36
4.8
ELECTRO-SWELLING
OF
T-SNARE/SYNTAXIN-1
GUVS
.
36
4.9
SUV/GUV
FUSION
ASSAY
.
37
4.10
SUV/GUV
DOCKING
ASSAY
.
38
4.10.1
MUNCL8-L
DOCKING
ASSAY
.
38
4.10.2
RAB3A/RIM1
DOCKING
ASSAY
.
39
4.11
CROSSLINKING
OF
MUNCL8-L
BPA
MUTANTS
.
39
4.12
WESTERN
BLOT
.
40
4.13
STATISTICAL
ANALYSIS
.
41
4.14
CONTRIBUTIONS
BY
OTHER
RESEARCHERS
.
41
5
RESULTS
.
42
5.1
RECONSTITUTED
LIPOSOME
FUSION
ASSAY
.
42
5.2
STAGE-SPECIFIC
EFFECTS
OF
MUNCL8-L
DEPEND
ON
THE
RECOGNITION
OF
THE
COGNATE
V-SNARE
43
5.3
MOLECULAR
DETAILS
OF
THE
VAMP2*MUNCL8-L
INTERACTION
.
45
5.3.1
SITE-SPECIFIC
PHOTO-CROSSLINKING
MAPS
THE
VAMP2-MUNCL8-1
BINDING
INTERFACE
IN
THE
RECONSTITUTED
TEMPLATE
COMPLEX
.
45
5.3.2
Q301
IN
HELIX
11
OF
MUNCL8-L
IS
ESSENTIAL
FOR
VAMP2
BINDING
.
48
5.3.3
THE
VAMP2-MUNCL8-1
INTERACTION
IS
REQUIRED
TO
PROMOTE
LIPID-MIXING
IN
RECONSTITUTED
MEMBRANE
FUSION
ASSAYS
.
50
5.4
MUNCL8-L
PHOSPHORYLATION
.
52
5.4.1
A
PHOSPHOMIMETIC
MUTATION
IN
HELIX
12
OF
MUNCL8-L
REGULATES
THE
TEMPLATING
FUNCTION
IN
A
STAGE-SPECIFIC
MANNER
.
52
5.4.2
MUNCL8-L
S345BPA
SHOWS
AN
INTRAMOLECULAR
CROSS-LINK
TO
THE
FURLED
LOOP
.
53
5.4.3
CONSTITUTIVELY
OPEN
MUND8-1
AND
SYNTAXIN-1
MUTANTS
COUNTERACT
THE
INHIBITORY
EFFECT
OF
THE
S345E
MUTATION
.
57
5.5
STXBP1
SYNDROME
.
60
5.5.1
MISSENSE
MUTATIONS
IN
MUNCL8-CAUSE
STXBP1
SYNDROME
.
61
5.5.2
ANALYSING
THE
EFFECTS
OF
MUNL8-1
MUTATIONS
ON
PROTEIN
STABILITY
.
61
5.5.3
THE
ANALYZED
MUNCL8-L
MUTANTS
DO
NOT
AGGREGATE
IN
VITRO
.
62
5.5.4
DIFFERENTIAL
EFFECTS
OF
MUNCL8-L
MUTATIONS
ON
SNARE
FUSION
.64
5.5.5
DISEASE-CAUSING
MUNCL8-L
MUTATIONS
CAUSE
STAGE-SPECIFIC
EFFECTS
.
66
5.5.6
PREDICTING
MUNCL8-L
MUTATION
SEVERITY
.
67
5.6
RECONSTITUTING
VESICLE
TETHERING
.
69
5.6.1
RAB3A
NUCLEOTIDE
EXCHANGE
AND
LIPID
MODIFICATION
.
69
5.6.2
RAB3A
AND
RIM1
PROMOTE
SUV-GUV
ASSOCIATION
IN
A
NUCLEOTIDE-SPECIFIC
MANNER
.
70
5.6.3
THE
RAB3A-RIM1
INTERACTION
STIMULATES
SNARE-MEDIATED
FUSION
.
71
II
6
DISCUSSION
.
74
6.1
MUNCL8-1*VAMP2
INTERACTIONS
ARE
ESSENTIAL
FOR
TEMPLATE
COMPLEX
FORMATION
.
74
6.2
PHOSPHORYLATION
OF
MUNC18-1
NEGATIVELY
INFLUENCES
THE
DYNAMICS
OF
HELIX12
AND
BLOCKS
FUSION
IN
A
STAGE-SPECIFIC
MANNER
.
76
6.3
WHEN
DOES
SNAP-25
ENTER
THE
STAGE?
.
78
6.4
MISSENSE
MUTATIONS
IN
MUNCL8-L
CAUSE
STXBPL-SYNDROME
.
80
6.4.1
DISEASE-CAUSING
MUNCL8-L
MUTATIONS
LEAD
TO
PROTEIN
INSTABILITY
.
80
6.4.2
THE
TESTED
DISEASE-CAUSING
MUTATIONS
DO
NOT
SHOW
DOMINANT
NEGATIVE
PROPERTIES
.
81
6.4.3
DISEASE-CAUSING
MUNCL8-L
MUTANTS
SHOW
IMPAIRED
STIMULATION
ON
SNARE-MEDIATED
FUSION
.
81
6.4.4
ALTERNATIVE
DISEASE
MECHANISMS
FOR
MUNCL8-L
T361I,
H445P,
AND
L446F.
82
6.4.5
IN
SILICO
MUTANT
PREDICTION
TO
CLASSIFY
STXBP1
MUTATIONS
.
84
6.5
RECONSTITUTING
SYNAPTIC
VESICLE
TETHERING
.
84
7
APPENDIX
.
87
7.1
LIST
OF
FIGURES
.
87
7.2
LIST
OF
TABLES
.
88
7.3
ABBREVIATIONS
.
88
7.4
LITERATURE
.
90
8
PUBLICATIONS
.
106
9
ACKNOWLEDGEMENTS.
.
107
III |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | André, Timon 1989- |
| author_GND | (DE-588)1160962685 |
| author_facet | André, Timon 1989- |
| author_role | aut |
| author_sort | André, Timon 1989- |
| author_variant | t a ta |
| building | Verbundindex |
| bvnumber | BV049564364 |
| ctrlnum | (OCoLC)1372152447 (DE-599)DNB1279866772 |
| discipline | Medizin |
| discipline_str_mv | Medizin |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV049564364 |
| illustrated | Illustrated |
| index_date | 2024-07-03T23:29:08Z |
| indexdate | 2024-07-10T10:10:48Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-034909706 |
| oclc_num | 1372152447 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | iii, 107 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2022 |
| publishDateSearch | 2022 |
| publishDateSort | 2022 |
| record_format | marc |
| spelling | André, Timon 1989- Verfasser (DE-588)1160962685 aut Roles of Munc18-1 in synaptic vesicle priming and disease presented by Timon Christian André (M.Sc.) Heidelberg 2022 iii, 107 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruprecht-Karls-University Heidelberg 2022 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1279866772/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034909706&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p dnb 20230215 DE-101 https://d-nb.info/provenance/plan#dnb 2\p dnb 20230215 DE-101 https://d-nb.info/provenance/plan#dnb |
| spellingShingle | André, Timon 1989- Roles of Munc18-1 in synaptic vesicle priming and disease |
| subject_GND | (DE-588)4113937-9 |
| title | Roles of Munc18-1 in synaptic vesicle priming and disease |
| title_auth | Roles of Munc18-1 in synaptic vesicle priming and disease |
| title_exact_search | Roles of Munc18-1 in synaptic vesicle priming and disease |
| title_exact_search_txtP | Roles of Munc18-1 in synaptic vesicle priming and disease |
| title_full | Roles of Munc18-1 in synaptic vesicle priming and disease presented by Timon Christian André (M.Sc.) |
| title_fullStr | Roles of Munc18-1 in synaptic vesicle priming and disease presented by Timon Christian André (M.Sc.) |
| title_full_unstemmed | Roles of Munc18-1 in synaptic vesicle priming and disease presented by Timon Christian André (M.Sc.) |
| title_short | Roles of Munc18-1 in synaptic vesicle priming and disease |
| title_sort | roles of munc18 1 in synaptic vesicle priming and disease |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1279866772/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034909706&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT andretimon rolesofmunc181insynapticvesiclepriminganddisease |
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