HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases: = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Volltext Volltext Inhaltsverzeichnis |
| Physical Description: | 83 S. Ill., graph. Darst. |
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Record in the Search Index
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|---|---|
| adam_text | HIV-1 NEF-ASSOCIATED
PAXILLIN AND PAK1/2
REGULATE ACTIVATION AND SECRETION
OF ADAM17/10 PROTEASES
HIV-1 NEF-ASSOZIIERTES PAXILLIN
UND PAK1/2
REGULIEREN DIE AKTIVIERUNG
UND SEKRETION
VON ADAM17/10 PROTEASEN
DER NATURWISSENSCHAFTLICHEN FAKULTAET
DER FRIEDRICH-ALEXANDER-UNIVERSITAET
ERLANGEN-NUERNBERG
ZUR
ERLANGUNG DES DOKTORGRADES DR.
RER. NAT
VORGELEGT VON
JUNG-HYUN LEE
AUS SEOUL, REPUBLIK KOREA
HTTP://D-NB.INFO/105363482X
TABLE OF CONTENTS
ABBREVIATIONS AND SYMBOLS
1. SUMMARY/ZUSAMMENFASSUNG 1
- ENGLISH/DEUTSCH
2. INTRODUCTION
3
2.1. THE HUMAN IMMUNODEFICIENCY VIRUS HIV-1 3
2.1.1. THE ROLE OF NEF IN HIV PATHOGENESIS 3
2.1.2. NEF AND BINDING PARTNERS 4
2.1.2.1. NEF AND NEF ASSOCIATED KINASE COMPLEX (NAKC) 4
2.1.2.2. NEF AND P21-ACTIVATED KINASE (PAK) 6
2.2. EXTRACELLULAR VESICLE, MICROVESICLE, EXOSOME 6
2.2.1. BIOGENESIS AND KEY FEATURES OF EXTRACELLULAR VESICLE 9
2.2.2. EXTRACELLULAR VESICLE AND DISEASE 13
2.3. ADAM PROTEASES 15
2.3.1. ADAM10 AND ADAM17 (TACE) 16
3. MATERIALS AND
METHODS 20
3.1. MATERIAL 20
3.1.1. CHEMICALS . 20
3.1.2. DNA MODIFYING ENZYMES 21
3.1.3. BUFFERS AND REAGENTS 21
3.1.3.1. GENERAL REAGENTS AND BUFFERS 21
3.1.3.2. BUFFER AND REAGENTS FOR SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS
(PAGE)
AND WESTERN BLOT ANALYSES 22
3.1.3.3. REAGENTS FOR CHEMICAL-COMPETENT E.COLI AND TRANSFORMATION BY
HEAT 23
3.1.3.4. BUFFERS AND REAGENTS FOR DNA ELECTROPHORESIS 23
3.1.3.5. LADDERS AND MARKERS 24
3.1.4. CELL CULTURE 24
3.1.4.1. CELL LINES AND CELL CULTURE MEDIA 24
3.1.4.2. ADDITIONAL CELL CULTURE MEDIA 25
3.1.4.3. ADDITIONAL MATERIAL 25
3.1.4.4. GENERAL CELL CULTURE REAGENTS 25
3.1.4.5. GENERAL CELL CULTURE DISHES AND PLASTIC WAERE 26
3.1.5. EQUIPMENTS AND INSTRUMENTS 26
3.1.6. SOFTWARE AND WEBSITES 27
3.1.7. ANTIBODIES USED FOR
WESTERN BLOT ANALYSES 28
3.1.8. DNA OLIGOS 29
3.1.8.1. PRIMERS.... 29
3.1.9. PLASMID VECTORS 30
3.1.9.1. DONATED OR PURCHASED VECTOR 30
3.1.9.2. CLONED VECTORS 30
3.2. METHODS 32
3.2.1. GENERAL MOLECULAR BIOLOGY METHODS 32
3.2.1.1. TRANSFORMATION BY HEAT 32
3.2.2. DNA BASED MOLECULAR BIOLOGY METHODS 32
3.2.2.1. ISOLATION OF DNA 32
3.2.2.2. NUCLEIC ACID QUANTIFICATION 33
3.2.2.3. SEPARATION OF DNA USING AGAROSE GEL ELECTROPHORESIS 33
3.2.2.4. POLYMERASE CHAIN REACTION (PCR) BASED METHODS 33
3.2.2.5. CLONING OF DNA FRAGMENTS OR PCR PRODUCTS 34
3.2.3. PROTEIN BIOCHEMISTRY METHODS 35
3.2.3.1. GENERATION OF CELL LYSATES 35
3.2.3.2. DISCONTINUOUS SDS-PAGE: LAEMMLI METHOD 35
3.2.3.3. IMMUNOPRECIPITATION 36
3.2.3.4. WESTERN BLOT ANALYSES 36
3.2.4. CELL CULTURE 37
3.2.4.1. CELL LINES 37
3.2.4.2. CRYOPRESERVATION OF CELL
LINES 37
3.2.4.3. HEAT INACTIVATION OF FETAL CALF SERUM (FCS) 37
3.2.5. TRANSIENT TRANSFECTION
METHODS 37
3.2.5.1 CALCIUM PHOSPHATE 37
3.2.5.2. LIPOFECTION OF DNA WITH LIPOFECTAMINE*
LTX AND PLUS* (LNVHROGEN)
REAGENT 38
3.2.6. ISOLATION AND PURIFICATION OF EXTRACELLULAR VESICLES
(EVS) 38
3.2.7. ISOLATION OF LIPID RAFTS VIA DISCONTINUOUS
SUCROSE DENSITY GRADIENT 39
4. TASKS
40
5. RESULTS
42
5.1. NEF AND NAKC ACTIVATE AND SECRET ADAM PROTEASE TACE (ADAM17) 42
5.1.1. NEF AND NAKC INDUCE THE ACTIVATION OF TACE 42
5.1.2. NEF/MNAKC TRANSFER TACE INTO EXTRAOELLULAR VESICTES (EVS) 43
5.2. PAXILLIN AND EED LINK NAKC TO TACE 45
5.2.1. NEF/NAKC PHOSPHORYLATE PAXILLIN AT Y118 RESIDUE 45
5.2.2. EED BRINGS
TACE TO NEF/NAKC AND PAXILLIN INTERACT WITH ACTIVATED
TACE 47
5.2.3. PAXILLIN MEDIATES TACE ACTIVATION AND SECRETION PROCESSES 48
5.3. PAK1 AND PAK2 REGLUATE PAXILLIN-TACE ASSOCIATION 49
5.3.1. PAXILLIN-TACE ASSOCIATION IS REGULATED BY PHOSPHORYLATION OF
PAXILLIN 49
5.3.2. PAK1 AND PAK2 INFLUENCE ON PAXILLIN AND TACE ASSOCIATION. 50
5.3.3. NEF/ NAKC MODULATE PAK1 AND PAK2 ACTIVITIES 53
5.4. PAK1 AND PAK2 REGULATE PAXILLIN-TACE SECRETION 54
5.4.1. PAK1 AND PAK2 REGULATE TRANSFER OF TACE INTO EVS 54
5.4.2. SECRETION OF TACE INTO LIPID RAFTS IS REGULATED
BY PAK KINASES 55
5.5 MELANOMA CELLS UPLOAD
ADAM
10 INTO EVS BY THE SAME MECHANISM AS HIV-1
NEF 56
5.5.1. MELANOMA CELLS ASSEMBLE THE SIMILAR SIGNALING COMPLEX 56
5.5.2. MELANOMA CELLS REGULATE ADAM10 TRANSFER INTO EVS THROUGH
PAXILLIN 58
5.5.3. PAK1 AND PAK2 REGULATE PAXILLIN AND ADAM10 ASSOCIATION 59
5.5.4. PAXILLIN SECRETION IS REGULATED BY PHOSPHORYLATION OF PAXILLIN BY
PAK1 AND
PAK2 60
5.6. PLASMA FROM HIV AND MELANOMA PATIENTS HARBORS ADAMS-CONTAINING EVS
61
6. DISCUSSION 63
6.1. PAXILLIN AND EED LINK NEF SIGNALING COMPLEX TO TACE 63
6.2. PHOSPHORYLATION OF PAXILLIN BY PAK1 AND PAK2 REGULATES
ADAM-CONTAINED EVS
SECRETION VIA LIPID RAFTS 67
6.3. PLASMA FROM HIV AND MELANOMA PATIENTS HARBORS ADAMS-CONTAINING EVS
69
6.4. SUMMARY 70
7. REFERENCES
72
|
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| spelling | Lee, Jung-hyun Verfasser aut HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen vorgelegt von Jung-Hyun Lee HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen 2014 83 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Erlangen-Nürnberg 2014 HIV-Infektion (DE-588)4203852-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content HIV-Infektion (DE-588)4203852-2 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:29-opus4-43545 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus4-43545 Resolvingsystem kostenfrei Volltext http://opus4.kobv.de/opus4-fau/frontdoor/index/index/docId/4354 Verlag kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027296553&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Lee, Jung-hyun HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen HIV-Infektion (DE-588)4203852-2 gnd |
| subject_GND | (DE-588)4203852-2 (DE-588)4113937-9 |
| title | HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen |
| title_alt | HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen |
| title_auth | HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen |
| title_exact_search | HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen |
| title_full | HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen vorgelegt von Jung-Hyun Lee |
| title_fullStr | HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen vorgelegt von Jung-Hyun Lee |
| title_full_unstemmed | HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen vorgelegt von Jung-Hyun Lee |
| title_short | HIV-1 Nef-associated Paxillin and Pak1/2 regulate activation and secretion of ADAM17/10 proteases |
| title_sort | hiv 1 nef associated paxillin and pak1 2 regulate activation and secretion of adam17 10 proteases hiv 1 nef assoziiertes paxillin und pak1 2 regulieren die aktivierung und sekretion von adam17 10 proteasen |
| title_sub | = HIV-1 Nef-assoziiertes Paxillin und Pak1/2 regulieren die Aktivierung und Sekretion von ADAM17/10 Proteasen |
| topic | HIV-Infektion (DE-588)4203852-2 gnd |
| topic_facet | HIV-Infektion Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:29-opus4-43545 http://opus4.kobv.de/opus4-fau/frontdoor/index/index/docId/4354 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027296553&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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