Role of the ubiquitin-like modifier FAT10 in protein degradation and ...

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Contents Acknowledgements 3 Summary / Zusammenfassung 4 English . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Deutsch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Introduction 9 Protein Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 The Ubiquitin-Proteasome-System . . . . . . . . . . . . . . . . . . . . . . . 10 The Proteasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 The 20S Proteasome . . . . . . . . . . . . . . . . . . . . . . . . 10 The 26S Proteasome . . . . . . . . . . . . . . . . . . . . . . . . 11 Ubiquitin and Ubiquitin-Like Proteins . . . . . . . . . . . . . . . . . . 14 Ubiquitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Ubiquitin-Like Proteins . . . . . . . . . . . . . . . . . . . . . . 18 Ubiquitin-Like Modifiers . . . . . . . . . . . . . . . . . . . . . . 18 FAT10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Ubiquitin Domain Proteins . . . . . . . . . . . . . . . . . . . . 24 NUB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 The Autophagy-Lysosome System . . . . . . . . . . . . . . . . . . . . . . . . 28 Misfolded Protein Stress, Aggresomes and the Connection between Au- tophagy and the UPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 HDAC6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Chapter 1: The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10 36 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Experimental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . 56 1
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 Chapter 2: Degradation of FAT10 by the 26S proteasome in vitro is indepen- dent of ubiquitylation but relies on NUB1L 60 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Chapter 3: The ubiquitin-like modifier FAT10 interacts with HDAC6 and lo- calizes to aggresomes under proteasome inhibition 77 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Chapter 4: FAT10-deficient mice display a prolonged CD8 + T-cell response after LCMV infection 104 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Chapter 5: Generation of mouse monoclonal antibodies directed against human FAT10 111 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Discussion 120 References 126 Abbreviations 142 Record of Contributions 145 2
Page 1: Role of the ubiquitin-like modifier
Page 5 and 6: Summary / Zusammenfassung English F
Page 7 and 8: Summary In conclusion, these result
Page 9 and 10: Summary nicht von der katalytischen
Page 11 and 12: The Ubiquitin-Proteasome-System The
Page 13 and 14: Introduction Figure 1: The 26S prot
Page 15 and 16: Introduction are the major source o
Page 17 and 18: Introduction majority of ubiquitin-
Page 19 and 20: Introduction example contained in t
Page 21 and 22: Introduction two modifiers apart fr
Page 23 and 24: Introduction Figure 4: Comparison o
Page 25 and 26: Introduction dependent apoptosis in
Page 27 and 28: Introduction Interestingly, a recen
Page 29 and 30: The Autophagy-Lysosome System Intro
Page 31 and 32: Introduction Figure 5: Molecular ma
Page 33 and 34: Introduction The transport of misfo
Page 35 and 36: Introduction where the proteasome i
Page 37 and 38: Chapter 1 The UBA domains of NUB1L
Page 39 and 40: Introduction Chapter 1 Ubiquitin, a
Page 41 and 42: Chapter 1 no correlation between th
Page 43 and 44: Chapter 1 covered proteins were sep
Page 45 and 46: Chapter 1 Figure 8: Accelerated deg
Page 47 and 48: Chapter 1 Treatment with IFN-γ and
Page 49 and 50: Chapter 1 ing SUMO-1-GFP or the C-t
Page 51 and 52: Chapter 1 Figure 12: Accelerated de
Page 53 and 54:
Chapter 1 Figure 13: Co-immunopreci
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Chapter 1 domains of NUB1L is requi
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Chapter 1 Rad23 (Verma et al., 2004
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Chapter 1 FAT10-GFP expressing vect
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Chapter 2 Degradation of FAT10 by t
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Introduction Chapter 2 The proteaso
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Chapter 2 From our previous data it
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Chapter 2 Figure 14: Purified compo
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Chapter 2 evaluation of the NUB1L b
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Chapter 2 teins which contain intri
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Chapter 2 Combined with our studies
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Chapter 2 Recombinant expression of
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Chapter 2 et al., 2003), both at a
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Abstract Chapter 3 During misfolded
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Chapter 3 tion between the UPS and
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Chapter 3 Figure 18: HDAC6 interact
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Chapter 3 Figure 19: Both the BUZ d
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Chapter 3 of HDAC6 was required for
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Chapter 3 precipitates with HDAC6 (
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90 Chapter 3
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Chapter 3 Figure 23: The model conj
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Chapter 3 HDAC6 is required for the
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Chapter 3 Figure 26: Both ubiquitin
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Chapter 3 also revealed endogenous,
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Chapter 3 and/or its conjugates is
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Chapter 3 (Hipp et al., 2005), pEGF
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Chapter 4 FAT10-deficient mice disp
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Chapter 4 Once the infection is cle
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Chapter 4 As both wild-type as well
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Chapter 4 Peptides. The synthetic p
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Introduction Chapter 5 Antibodies a
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Chapter 5 binant human and mouse GS
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Chapter 5 Figure 34: The antibodies
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Chapter 5 Figure 36: The antibody s
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Discussion 30 years ago, ubiquitin
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Discussion monomeric ubiquitin as w
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Discussion Alternatively, FAT10 cou
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References D. T. Akey, X. Zhu, M. D
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References Y.-H. Chiu, Q. Sun, and
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References S. Gunawardena, L.-S. He
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References J. A. Johnston, M. E. Il
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References F. Lévy, N. Johnsson, T
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References J.-H. Park, H.-S. Jong,
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References P. Schreiner, X. Chen, K
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References S. Vijay-Kumar, C. E. Bu
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Abbreviations aa amino acid AAA ATP
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Amino Acids Alanine Ala A Arginine
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