Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Münster
2016
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| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | VII, 173 Blätter Illustrationen, Diagramme 30 cm |
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| 245 | 1 | 0 | |a Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica |c vorgelegt von Benjamin Andrè Grabowski |
| 264 | 1 | |a Münster |c 2016 | |
| 300 | |a VII, 173 Blätter |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 Westfälischen Wilhelms-Universität Münster |d 2016 |g Beschränkt für den Austausch | ||
| 650 | 0 | 7 | |a Proteine |0 (DE-588)4076388-2 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Yersinia enterocolitica |0 (DE-588)4287241-8 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Translokation |0 (DE-588)4185910-8 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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Datensatz im Suchindex
| _version_ | 1804177025813970944 |
|---|---|
| adam_text | TABLE OF CONTENTS
ABSTRACT.......................................................................................................................
I
TABLE OF
CONTENTS........................................................................................................
ILL
1
INTRODUCTION..........................................................................................................
1
1.1 BACTERIA-DERIVED THERAPEUTICS - THE *DRUGS FROM BUGS* CONCEPT
..................
1
1.1.1 MECHANISMS OF IMMUNOMODULATION BY HUMAN PATHOGENS
................
1
1.1.1.1 HOST RESPONSES TO FOREIGN ORGANISMS
................................
1
1.1.1.2 MICROBIAL RESPONSES TO IMMUNE REACTIONS........................
2
1.1.2 CELL-PENETRATING PEPTIDES AND PROTEINS
...........................................
4
1.1.2.1 DEFINITION, CLASSES AND FEATURES OF CELL-PENETRATING
PEPTIDES..............................................................................
4
1.1.2.2 MECHANISMS OF TRANSLOCATION
..............................................
6
1.1.2.3 CELL-PENETRATING EFFECTOR PROTEINS (CPES)
...........................
9
1.2 YERSINIA ENTEROCOLITICA - ORIGIN AND
PATHOGENICITY......................................... 10
1.3 YERSINIA OUTER PROTEINS (YOPS)
......................................................................
11
1.3.1 YOPM - A CELL-PENETRATING SCAFFOLD PROTEIN
......................................
12
1.3.2 YOPO - A MULTIDOMAIN EFFECTOR PROTEIN
...........................................
14
1.3.3 YOPE - A GTPASE ACTIVATING PROTEIN
...............................................
14
1.3.3.1 STRUCTURE AND
FUNCTION......................................................... 14
1.3.3.2 POTENTIAL THERAPEUTIC USES OF CELL-PENETRATING RYOPE......... 16
1.3.4 YOPT - AN IRREVERSIBLE INHIBITOR OF
RHOGTPASES.............................. 16
1.3.4.1 STRUCTURE AND
FUNCTION......................................................... 16
1.3.4.2 POTENTIAL THERAPEUTIC USES OF CELL-PENETRATING RYOPT......... 17
1.3.5 YOPP - A HIGHLY POTENT ANTI-INFLAMMATORY EFFECTOR PROTEIN
...............
18
1.3.5.1 STRUCTURE AND
FUNCTION......................................................... 18
1.3.5.2 POTENTIAL THERAPEUTIC USES OF CELL-PENETRATING RYOPP......... 21
1.3.6 YOPH - A VERSATILE PHOSPHOTYROSINE PHOSPHATASE
...........................
22
1.3.6.1 STRUCTURE AND
FUNCTION......................................................... 22
1.3.6.2 POTENTIAL THERAPEUTIC USES OF CELL-PENETRATING RYOPH......... 26
1.4
OBJECTIVES......................................................................................................
29
2 MATERIAL
................................................................................................................
30
2.1 OLIGONUCLEOTIDES &
PLASMIDS........................................................................
30
2.1.1
OLIGONUCLEOTIDES...............................................................................
30
2.1.2
PLASMIDS...........................................................................................
32
2.2 BIOLOGICAL
MATERIALS........................................................................................
35
2.2.1 BACTERIAL STRAINS
................................................................................
35
2.2.2 EUKARYOTIC CELL
LINES...........................................................................
35
2.3 MEDIA & BUFFER
SOLUTIONS................................................................................
36
2.3.1 BACTERIAL GROWTH
MEDIA......................................................................
36
2.3.2 EUKARYOTIC CELL LINES
..........................................................................
36
2.3.3 COMMON BUFFERS AND SOLUTIONS
..........................................................
37
2.4 CHEMICALS, BUFFERS AND
DYES.........................................................................
37
2.5
ENZYMES........................................................................................................
38
2.6 SIZE
STANDARDS..............................................................................................
39
2.7
ANTIBODIES......................................................................................................
39
2.7.1 PRIMARY
ANTIBODIES............................................................................
39
2.7.2 SECONDARY
ANTIBODIES.......................................................................
40
2.8 K
ITS.................................................................................................................
40
2.9 LABORATORY EQUIPMENT
...................................................................................
41
3
METHODS................................................................................................................
43
3.1 MOLECULAR BIOLOGY
METHODS...........................................................................
43
3.1.1 PLASMID
ISOLATION...............................................................................
43
3.1.2 NUCLEIC ACID
QUANTIFICATION................................................................
43
3.1.3 POLYMERASE CHAIN
REACTION................................................................
43
3.1.4 AGAROSE GEL ELECTROPHORESIS AND ISOLATION OF SINGLE AMPLICONS
.......
44
3.1.5
CLONING.............................................................................................
45
3.1.5.1 RESTRICTION BASED (*CLASSICAL*)
CLONING................................. 45
3.1.5.2 RESTRICTION-FREE CLONING
......................................................
46
3.1.5.3 SITE-DIRECTED
MUTAGENESIS.................................................. 46
3.1.6 COLONY PCR AND
SEQUENCING........................................................... 47
3.1.7 IN VITRO PHOSPHATASE
ASSAY............................................................... 47
3.1.8 QUANTITATIVE REAL-TIME PCR
(QPCR).................................................. 46
3.1.8.1 RNA
ISOLATION.....................................................................
46
3.1.8.2 REVERSE
TRANSCRIPTION.........................................................
48
3.1.8.3 QUANTITATIVE REAL-TIME PC R
................................................
49
3.2 MICROBIOLOGY METHODS
...................................................................................
49
3.2.1 CULTURING AND CRYOCONSERVATION OF BACTERIAL
STRAINS........................... 49
3.2.2 DETERMINING THE OPTICAL DENSITY OF BACTERIAL CULTURES
.........................
50
3.2.3 TRANSFORMATION OF BACTERIAL CELLS
......................................................
50
3.2.3.1 GENERATING CHEMICAL COMPETENT E. C O LI
.............................
51
3.2.3 2 TRANSFORMING CHEMICAL COMPETENT E. C O LI
........................
51
3.2.3.3 GENERATING ELECTRO-COMPETENT Y. ENTEROCOLITICA
................
51
3.2.3.4 TRANSFORMING ELECTRO-COMPETENT Y. ENTEROCOLITICA
.............
51
3.2.4 EXPRESSION OF RECOMBINANT PROTEINS
................................................
52
3.2.4.1 INDUCTION ASSAY
..................................................................
52
3 2.4.2 LARGE SCALE PROTEIN
EXPRESSION......................................... 53
3.3 PROTEIN BIOCHEMISTRY
METHODS.......................................................................
53
3.3.1 PROTEIN
PURIFICATION............................................................................
53
3.3.2 PROTEIN
QUANTIFICATION.........................................................................
54
3.3.2.1 BICINCHONINIC ACID (BCA) ASSAY
........................................
54
3.3.2.2
SDS-PAGE........................................................................
55
3.3.3 PROTEIN LABELING WITH FLUORESCENT DYES
..............................................
55
3.3.3.1 FLUORESCEIN-ISOTHIOCYANATE (FITC)
....................................
55
3.3 3.2 5-(AND-6)-CARBOXYNAPHTHOFLUORESCEIN (N F )
.......................
56
3.3.4 SODIUM DODECYL SULFATE POLY ACRYLAMIDE GEL ELECTROPHORESIS (SDS-
PAGE)
..............................................................................................
56
3.3.4.1 GEL PREPARATION
..................................................................
57
3.3.4.2 ELECTROPHORESIS
..................................................................
58
3 3.4.3 COOMASSIE BLUE
STAINING.................................................... 58
3 3.4.4 WESTERN BLOTTING
.................................................................
59
3.3.5 PULLDOWN
EXPERIMENTS.......................................................................
60
3.4 CELL BIOLOGY METHODS
....................................................................................
62
3.4.1 CULTURING AND CRYOCONSERVATION OF EUKARYOTIC CELLS
..........................
62
3.4.1.1 CULTURING OF HELA CELLS
.....................................................
62
3.4.1.2 CULTURING OF THP-1 AND JURKAT CELLS
................................
62
3.4.2 CELL
FRACTIONATION...............................................................................
63
3.4.3 FLUORESCENCE ACTIVATED CELL SCANNING
(FACS).................................. 64
3.4.3.1 CELLULAR UPTAKE KINETICS - FITC-LABELED PROTEINS
..............
64
3.4 3.2 CELLULAR UPTAKE KINETICS - NF-LABELED PROTEINS
.................
65
3.4.3.3 CO-INCUBATION WITH ENDOCYTOSIS INHIBITORS
.........................
66
3.4.3.4 EGF-RECEPTOR INTERNALIZATION
..............................................
66
3.4.4 FLUORESCENCE MICROSCOPY
.................................................................
67
3.4.5 TRANSMISSION ELECTRON
MICROSCOPY................................................... 68
3.4.6 CYTOTOXICITY TEST - LACTATE DEHYDROGENASE (LDH) RELEASE ASSAY
......
68
3.4.7 PROLIFERATION TEST - BROMODEOXYURIDINE (BRDU) INCORPORATION ASSAY
69
3.4.8
TRANSFECTION......................................................................................
70
3.4.9 STIMULATION
ASSAYS............................................................................
70
3.4.9.1 TYROSINE
PHOSPHORYLATION................................................... 70
3 4.9.2 CYTOKINE PRODUCTION IN THP-1
CELLS.................................... 71
3.4.10 GAP CLOSURE MIGRATION ASSAY (*WOUND HEALING ASSAY*)
...................
72
3.4.11
INFECTION...........................................................................................
73
3.4.11.1 PHOSPHOTYROSINE REDUCTION ASSAY
....................................
73
3.4.11.2 INVASION ASSAY
.................................................................
73
3.4.11.3 IL-8 SECRETION
ASSAY......................................................... 74
3.5 COMPUTER BASED
METHODS..............................................................................
74
3.5.1 PREDICTION OF PUTATIVE PROTEIN TRANSDUCTION DOMAINS
........................
74
3.5.2 SEQUENCE AND STRUCTURE ANALYSIS, ALIGNMENTS AND PRIMER DESIGN ....
75
3.5.3 OTHER COMPUTER BASED
METHODS........................................................ 75
4
RESULTS.................................................................................................................
76
4.1 CLONING AND EXPRESSION OF RECOMBINANT YERSINIA OUTER PROTEINS (YOPS)
.......
76
4.2 ANALYSIS OF THE CELL-PENETRATING POTENTIAL OF
RYOPE........................................ 77
4.3 ANALYSIS OF THE CELL-PENETRATING POTENTIAL OF RYOPT
........................................
78
4.4 ANALYSIS OF THE CELL-PENETRATING POTENTIAL OF RYOPP
........................................
61
4.5 ANALYSIS OF THE CELL-PENETRATING AND THERAPEUTIC POTENTIAL OF RYOPH
.............
83
4.5.1 IN SILICO ANALYSIS AND EXPERIMENTAL VALIDATION OF CELL-PENETRATION
BY
RYOPH................................................................................................
83
4.5.2 DETERMINATION OF THE TRANSLOCATION ROUTE OF RYOPH
...........................
85
4.5.2.1 ENDOCYTOSIS VERSUS DIRECT PENETRATION
................................
85
4.5.2.2 CO-LOCALIZATION WITH ENDO-LYSOSOMAL MARKERS
...................
86
4.5.2.3 ENDOSOMAL
ESCAPE............................................................ 88
4.5.3 VALIDATION OF THE PREDICTED PROTEIN TRANSDUCTION DOMAIN
..................
91
4.5.4 CONSTRUCTION AND ANALYSIS OF YOPH FUSION PROTEINS FOR ENHANCED
CYTOSOLIC
DELIVERY..............................................................................
94
4.5.5 IN VITRO FUNCTIONALITY OF RYOPH AND ITS DERIVATIVES
.............................
97
4.5.5.1 PURIFICATION AND ENZYMATIC ACTIVITY
.....................................
97
4.5.5 2
CYTOTOXICITY.........................................................................
99
4.5.6 INTRACELLULAR FUNCTIONALITY OF RYOPH AND ITS DERIVATIVES IN HELA
CELLS 101
4.5.6.1 REDUCTION OF PHOSPHOTYROSINE
LEVELS.................................. 101
4.5.6.2 TIME-DEPENDENCY OF INTRACELLULAR RYOPH ACTIVITY
................
104
4.5.6 3 INHIBITION OF SPECIFIC SIGNALING PATHWAYS
............................
105
4.5.7 IMPACT OF RYOPH ON FOCAL ADHESIONS
................................................
107
4.5.7.1 DISRUPTION OF FOCAL ADHESION
COMPLEXES............................. 107
4.5.7.2 INTERFERENCE WITH CELL MIGRATION
..........................................
109
4.5.8 COMPLEMENTATION OF YOPH-DEFICIENT YERSINIA BY
RYOPH................... 110
4.5.6.1 INHIBITION OF
PHAGOCYTOSIS........................................... 110
4.5.5.2 INHIBITION OF IL-8 SECRETION
..................................................
112
4.5.9 INTRACELLULAR FUNCTIONALITY OF RYOPH AND ITS DERIVATIVES IN
LYMPHOCYTES......................................................................................
113
4.5.9.1 REDUCTION OF PHOSPHOTYROSINE LEVELS
.................................
113
4.5 9.2 INTERFERENCE WITH CELL PROLIFERATION
....................................
115
4.5.9.3 DOWN-REGULATION OF PRO-INFLAMMATORY CYTOKINES.................
116
4.6 PROTEIN KINASE C 5 - A NOVEL SUBSTRATE OF YOPH?
.........................................
118
5
DISCUSSION............................................................................................................
120
5.1 INCIDENCE OF CELL-PENETRATION AMONG YERSINIA OUTER
PROTEINS........................ 120
5.2 YERSINIA OUTER PROTEINS AS POTENTIAL THERAPEUTIC
AGENTS................................ 122
5.3 UPTAKE OF RYOPH INTO HOST CELLS
....................................................................
123
5.3.1 RYOPH IS TAKEN UP INTO DIFFERENT CELL TYPES
........................................
123
5.3.2 RYOPH IS TAKEN UP VIA ENDOCYTIC
PROCESSES...................................... 124
5.3.3 RYOPH DOES NOT HARBOR A CLASSICAL PROTEIN TRANSDUCTION
DOMAIN...... 126
5.3.4 ADDITIONAL PTDS DO NOT INCREASE THE UPTAKE EFFICIENCY OF RYOPH
.....
129
5.4 INTRACELLULAR FUNCTIONALITY OF
RYOPH.................................................................
130
5.4.1 RYOPH REDUCES INTRACELLULAR PHOSPHOTYROSINE LEVELS BUT DOES NOT
ELICIT EXPECTED DOWNSTREAM
EFFECTS.................................................. 130
5.4.2 RYOPH MIGHT NOT REACH ITS INTRACELLULAR TARGETS IN SUFFICIENT
AMOUNTS 134
5.5 INTERACTION OF RYOPH WITH PROTEIN KINASE C 5
................................................. 136
6 SUMMARY AND PERSPECTIVES
...............................................................................
138
7
REFERENCES...........................................................................................................
141
8
APPENDIX...............................................................................................................
164
8.1 ADDITIONAL FIGURES AND
TABLES.........................................................................
164
8.2
ABBREVIATIONS.................................................................................................
169
8.3 LIST OF
FIGURES..................................................................................................
171
8.4 LIST OF
TABLES...................................................................................................
173
|
| any_adam_object | 1 |
| author | Grabowski, Benjamin 1987- |
| author_GND | (DE-588)1119747775 |
| author_facet | Grabowski, Benjamin 1987- |
| author_role | aut |
| author_sort | Grabowski, Benjamin 1987- |
| author_variant | b g bg |
| building | Verbundindex |
| bvnumber | BV044025632 |
| ctrlnum | (OCoLC)964574300 (DE-599)DNB1120955718 |
| dewey-full | 610 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV044025632 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T07:41:31Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-029433030 |
| oclc_num | 964574300 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | VII, 173 Blätter Illustrationen, Diagramme 30 cm |
| publishDate | 2016 |
| publishDateSearch | 2016 |
| publishDateSort | 2016 |
| record_format | marc |
| spelling | Grabowski, Benjamin 1987- Verfasser (DE-588)1119747775 aut Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica vorgelegt von Benjamin Andrè Grabowski Münster 2016 VII, 173 Blätter Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Westfälischen Wilhelms-Universität Münster 2016 Beschränkt für den Austausch Proteine (DE-588)4076388-2 gnd rswk-swf Yersinia enterocolitica (DE-588)4287241-8 gnd rswk-swf Translokation (DE-588)4185910-8 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Yersinia enterocolitica (DE-588)4287241-8 s Proteine (DE-588)4076388-2 s Translokation (DE-588)4185910-8 s DE-604 B:DE-101 application/pdf http://d-nb.info/1120955718/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029433030&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Grabowski, Benjamin 1987- Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica Proteine (DE-588)4076388-2 gnd Yersinia enterocolitica (DE-588)4287241-8 gnd Translokation (DE-588)4185910-8 gnd |
| subject_GND | (DE-588)4076388-2 (DE-588)4287241-8 (DE-588)4185910-8 (DE-588)4113937-9 |
| title | Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica |
| title_auth | Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica |
| title_exact_search | Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica |
| title_full | Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica vorgelegt von Benjamin Andrè Grabowski |
| title_fullStr | Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica vorgelegt von Benjamin Andrè Grabowski |
| title_full_unstemmed | Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica vorgelegt von Benjamin Andrè Grabowski |
| title_short | Novel cell-penetrating effector proteins for therapeutic applications derived from Yersinia enterocolitica |
| title_sort | novel cell penetrating effector proteins for therapeutic applications derived from yersinia enterocolitica |
| topic | Proteine (DE-588)4076388-2 gnd Yersinia enterocolitica (DE-588)4287241-8 gnd Translokation (DE-588)4185910-8 gnd |
| topic_facet | Proteine Yersinia enterocolitica Translokation Hochschulschrift |
| url | http://d-nb.info/1120955718/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029433030&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT grabowskibenjamin novelcellpenetratingeffectorproteinsfortherapeuticapplicationsderivedfromyersiniaenterocolitica |
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