Molecular functions of the ubiquitin domain protein Herp in Synoviolin mediated endoplasmic reticulum associated protein degradation (ERAD) [Elektronische Ressource] / von Melanie Kny

Melanie Kny

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 6
• Langue: English

Extrait

Molecular functions of the ubiquitin domain protein Herp in
Synoviolin mediated endoplasmic reticulum associated protein
degradation (ERAD)

Dissertation

zur Erlangung des akademischen Grades

doctor rerum naturalium (Dr. rer. nat.)

im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Diplom-Ernährungswissenschaftlerin Melanie Kny


Präsident der Humboldt-Universität zu Berlin:
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I:
Prof. Dr. Lutz-Helmut Schön
Gutachter:
1. Professor Dr. Peter Michael Kloetzel
2. Professor Dr. Wolfgang Lockau
3. Professor Dr. Wolfgang Dubiel
Tag der mündlichen Prüfung: 23.06.2010Table of contents 2
Table of contents
ABSTRACT ........................................................................................................................................4
ZUSAMMENFASSUNG ......................................................................................................................5
1 INTRODUCTION.........................6
1.1 The ubiquitin proteasome system (UPS)..........................................................................6
1.1.1 Composition of the 26S proteasome...............7
1.1.2 The role of ubiquitin........................................7
1.1.3 The process of ubiquitination..........................................................8
1.1.4 E3 ubiquitin protein ligases...........................10
1.1.5 Deubiquitinating enzymes (DUBs) ................................................11
1.2 Endoplasmic reticulum (ER) protein quality control......................13
1.2.1 Protein synthesis at the ER..........................................................13
1.2.2 ER quality control, ER stress and the unfolded protein response (UPR)........................13
1.2.3 ER stress signalling......................................................................14
1.2.4 ER associated protein degradation (ERAD)..16
1.2.5 Synoviolin based ERAD complexes..............................................19
1.2.6 Homocysteine inducible endoplasmic reticulum - resident protein (Herp) ......................22
1.3 Aim of this study..............................................................................25
2 MATERIAL AND METHODS.....................................26
2.1 Instruments, consumables and chemicals.....................................26
2.2 Molecular biology ............................................28
2.2.1 Cultivation and storage of Escherichia coli (E.coli)........................................................28
2.2.2 Isolation of plasmid DNA from E.coli.............................................28
2.2.3 Separation of DNA fragments by agarose gel electrophoresis.......................................29
2.2.4 Determination of DNA and RNA concentration in solution.............29
2.2.5 Preparation of competent E.coli cells............30
2.2.6 Transformation of E.coli with plasmid DNA...................................30
2.2.7 Isolation of total RNA from HeLa cells..........................................30
2.2.8 Amplification of DNA by polymerase chain reaction (PCR)............31
2.2.9 Semiquantitative analysis of mRNA levels by reverse transcriptase (RT) PCR..............31
2.2.10 In vitro recombination of DNA...................................................32
2.2.11 Site-directed mutagenesis........................................................34
2.3 Tissue culture ..................................................35
2.3.1 Cell lines and media used in this study .........................................................................35
2.3.2 Culture of cells.............36
2.3.3 Cryoconservation and thawing of cells..........36
2.3.4 Transfection of mammalian cells..................37
2.3.5 Generation of a stable inducible cell line expressing Herp specific shRNA....................39
2.3.6 Cycloheximide chase analysis......................................................................................40
35
2.3.7 Metabolic labelling using [ S]-methionine/-cysteine and pulse chase analysis..............40
2.4 Protein biochemistry .......................................................................................................41
2.4.1 Lysis of mammalian cells..............................41
2.4.2 Determination of protein concentration in solution.........................................................41
2.4.3 Precipitation of proteins using trichloro acetic acid (TCA)..............41
2.4.4 Sodium dodecyl sulfate polyacrylamid gel electrophoresis (SDS-PAGE).......................42
2.4.5 Western blot analysis ...................................................................43
2.4.6 Protein visualisation.....................................43
2.4.7 Affinity precipitation of proteins.....................................................................................45
2.4.8 Separation of proteins by glycerol gradient centrifugation.............47
Table of contents 3
2.4.9 In vitro binding studies..................................................................................................47
2.5 Bioinformatics and databases........................49
3 RESULTS .................................................................50
3.1 The importance of the dynamics of Herp for ERAD.......................................................50
3.1.1 Herp is exchanged at Synoviolin based complexes50
3.1.2 Synoviolin complex components are not essential for the degradation of Herp..............52
3.1.3 Herp-K61R is stabilised and impairs the degradation of NHK........................................54
3.2 The role of Herp in maintaining the integrity of Synoviolin based complexes .............57
3.2.1 Herp does not alter the formation of Synoviolin oligomers57
3.2.2 Usp7 is a target of the Herp UBL domain......................................................................58
3.2.2.1 The AXXS motif contributes to an efficient binding of Usp7 to Herp ......................59
3.2.2.2 Herp recruits Usp7 to Synoviolin..........61
3.2.2.3 Usp7 does not affect the stability of Herp or NHK.................................................64
3.2.2.4 Herp is not involved in the regulation of p53.........................66
3.2.3 Ancient ubiquitous protein 1 (AUP1) is associated with Synoviolin................................69
3.2.3.1 Herp regulates the association of AUP1 with Synoviolin.......69
3.2.3.2 AUP1 binds to Synoviolin.....................................................................................70
3.2.3.3 AUP1 is required for the degradation of NHK.......................71
3.2.3.4 The AUP1-CUE domain is required for the efficient degradation of NHK...............72
3.3 Characterisation of Herp2 ...............................................................................................74
3.3.1 Herp2 reveals dynamics different from Herp.................................74
3.3.2 Herp and Herp2 form homo- and heterooligomers........................76
3.3.3 Herp2 is associated with Synoviolin based complexes..................................................78
4 DISCUSSION............................................................................................80
4.1 The role of the dynamics of Herp in ERAD.....................................................................80
4.1.1 The turnover of Herp at Synoviolin based ERAD complexes.........80
4.1.2 Correlation of the turnover of Herp and ERAD substrates.............83
4.2 The importance of Herp for the integrity of Synoviolin based complexes....................85
4.2.1 The impact of Herp on Synoviolin oligomerisation.........................................................85
4.2.2 Herp dependent recruitment of Usp7 to Synoviolin.......................87
4.2.3 Her association of Synoviolin and AUP1...................92
4.3 Comparison of Herp and Herp2 ......................................................95
4.4 Conclusion.......................................................................................97
LITERATURE...................................................................98
APPENDIX .....................................................................................................105
ABBREVIATIONS...........................................................105
DANKSAGUNG ..............................................................................................109
Abstract 4
Abstract
The accumulation of aberrant proteins in the endoplasmic reticulum (ER) induces the
unfolded protein response (UPR) pathway for surmounting this cellular stress situation. One
of the strongly UPR-induced genes in mammalia encodes the ubiquitin domain protein Herp.
Herp interacts with the E3 ligase Synoviolin, a central component of ER associated protein
degradation (ERAD) mediating multiprotein complexes. Dependent on its ubiquitin-like (UBL)
domain, Herp is required for the efficient degradation of Synoviolin substrates. The molecular
mechanism underlying this function of Herp is poorly understood.
In the present study, it was shown that Herp is continuously exchanged at Synov