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

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Acknowledgements First of all, I would like to thank Prof. Dr. Marcus Groettrup for giving me the opportunity to do this work and for teaching me a lot during these past few years. Many thanks also go to... Dr. Gunter Schmidtke for his biochemistry toolbox and for teaching me the “appropriate” way to ask questions. My fellow (PhD) students on the FAT10 project, expecially Sebastian Lukasiak, Anja Holtz and Christiane Peltzer, for many stimulating discussions and many bars of chocolate. Dr. Matthias Langhorst for taking the time to teach me confocal microscopy, Dr. (in spe) Jaqueline Möbius for teaching me how to do an ICS and Dr. Eva Schlosser for showing me how to make BMDCs. All of my colleagues in the Groettrup lab (whose names could not be cited due to space constraints) for always lending a hand when I needed it and for generally making this an enjoyable place to work in. Dr. Henner Brinkmann for helping me take my first steps in the exciting world of scientific research. Dr. Marian Schwartz for showing me how much fun biology could be. 1000 Dank auch den “Lustigen Biologen”, insbesondere Drs. Isa Pochic und Katrin Styp von Rekowski sowie Drs. (in spe) Eva Billerbeck, Helgard Fischer, Sonja Fraas, Nina Jagmann, Silke Litzinger und Sonja Weinitschke für die unzähligen gemeinsamen Stunden und die vielen Blicke über den Tellerrand hinaus. Last but not least gilt mein Dank meinen Eltern für ihre Liebe, ihre Unterstützung – und die frühkindliche Prägung auf die wissenschaftliche Denkweise. 3
Summary / Zusammenfassung English FAT10 is a ubiquitin-like protein which is encoded in the major histocompatibil- ity complex class I locus and is synergistically inducible with the proinflamma- tory cytokines IFN-γ and TNF-α in cells of nearly every tissue origin. It consists of two ubiquitin-like domains, which are connected by a short linker, and bears a free diglycine-motif at its C-terminus through which it can become covalently conjugated to so far unidentified target proteins. Overexpression of the wild-type protein – but not a diglycine-deficient mutant – leads to the induction of caspase- dependent apoptosis in murine as well as human cell lines. Both FAT10 and its conjugates are rapidly degraded by the proteasome at a similar rate, and the N- terminal fusion of FAT10 to long-lived proteins such as GFP or DHFR reduces their half-life as potently as their fusion with ubiquitin. The proteasomal degradation of FAT10, which occurs independently of ubiquitylation, can further be accelerated by non-covalent interaction with the UBL-UBA domain protein NEDD8 ultimate buster 1-long (NUB1L). The primary purpose of this thesis was to gain a more detailed insight into the mechanistics of FAT10-mediated protein degradation. Furthermore, a monoclonal antibody directed against human FAT10 was raised and FAT10 could be shown to be required for the proper down-regulation of the antiviral CD8 + T cell response. NUB1L, which was identified through a yeast two-hybrid screen as a non-covalent binding partner of FAT10, could previously be shown to accelerate the proteasomal degradation of FAT10 when the two proteins were co-expressed. This thesis dissects the molecular determinants of the aforementioned acceleration and un- covers a dual role of NUB1L as a proteasomal receptor for FAT10 as well as a facilitator of its degradation. Although FAT10 is capable of direct interaction with the 26S proteasome, NUB1L can function as a soluble FAT10-receptor by 4
Page 1 and 2: Role of the ubiquitin-like modifier
Page 3: Acknowledgements . . . . . . . . .
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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