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

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Summary binding to the proteasome with its UBL domain and to FAT10 with its UBA domains. Interestingly, the interaction of FAT10 with NUB1L is, at least in vivo, strictly dependent on the presence of all three of its UBA domains. The facilita- tion of FAT10 degradation, on the other hand, is strictly dependent on the abil- ity of NUB1L to interact with the 26S proteasome through its UBL domain and shows no requirement for any of its three UBA domains. Furthermore, degradation of the N-terminal UBL domain of FAT10 – which can interact with the 26S proteasome, but not with NUB1L – can still be accelerated by co-expression of NUB1L. Through experiments using an in vitro system consisting of purified 26S proteasome and recombinantly expressed substrates, this thesis demonstrates the 26S proteasome to be able to degrade the model substrate FAT10-dihydrofolate reduc- tase (DHFR) – although only in the presence of NUB1L. Furthermore, the study of FAT10-DHFR degradation in NUB1L knock-down cells revealed this absolute requirement for NUB1L to also apply to in vivo situations, suggesting that – at least in the case of this model substrate – mere binding of FAT10 to the proteasome is not sufficient to ensure the degradation of its target proteins. Another non-covalent interaction partner of FAT10, histone deacetylase 6 (HDAC6), was also identified through a yeast two-hybrid screen. HDAC6, which is a primarily cytoplasmatic protein, is involved in the deactylation of α-tubulin, HSP90 and cortactin. In addition to its two deacetylase domains, it also encom- passes a dynein-binding domain as well as a ubiquitn-binding zinc finger (BUZ domain) and tethers polyubiquitylated proteins to the dynein-motor during their microtubule-dependent transport to the aggresome. The herein presented results uncover a role for HDAC6 not only in the transport of polyubiquitylated cargo, but also in the transport of the ubiquitin-like modifier FAT10 and its conjugates. Under conditions of misfolded protein stress FAT10 interacts with HDAC6 and localizes to the aggresome in a microtubule-dependent manner. The binding of FAT10 is mediated by two different domains of HDAC6, the BUZ domain and surprisingly also the first catalytic domain, but it is not dependent on the catalytic activity of HDAC6. Furthermore, this thesis showed FAT10-containing as well as ubiquitin-containing aggresomes to be reduced in both size and number in HDAC6 deficient fibroblasts, which suggests that, although HDAC6 plays an important role in aggresomal targeting, it is not essential for the transport of proteasomal substrates to the aggresome. 5
Summary In conclusion, these results suggest FAT10 to mediate the rapid and inducible destruction of its target proteins through association with two different adaptor proteins. With help of the UBL-UBA domain protein NUB1L, FAT10 is able to promote the degradation of its conjugates by the 26S proteasome. Under conditions of proteasome impairment, however, FAT10 interacts with the linker protein HDAC6 and instead mediates the sequestration of its substrates in the aggresome. Deutsch FAT10 ist ein Ubiquitin-ähnliches Protein, welches im Haupt-Gewebe- Kompatibilitäts (MHC) Lokus der Klasse I kodiert liegt. Mit den proinflam- matorischen Zytokinen TNF-α und IFN-γ ist es in Zellen fast jeder Herkunft induzierbar. FAT10 besteht aus zwei durch einen kurzen Linker verbundenen Ubiquitin-ähnlichen Domänen und besitzt an seinem C-terminus ein Di-Glycin- Motiv, über welches es an seine Zielproteine kovalent konjugiert werden kann. Überexpression des wild-typ Proteins – aber nicht einer Mutante ohne Di-Glycin- Motiv – führt zur Induktion von Caspase-abhängiger Apoptose in murinen und humanen Zelllinien. Sowohl FAT10 als auch seine Konjugate werden vom Protea- som mit der gleichen Geschwindigkeit abgebaut; und die N-terminale Fusion von FAT10 an langlebige Proteine wie GFP oder DHFR reduziert deren Halbwertszeit ebenso potent wie eine Fusion mit Ubiquitin. Der proteasomale Abbau von FAT10 – welcher unabhängig von Ubiquitin erfolgt – kann zusätzlich beschleunigt werden durch nicht-kovalente Interaktion von FAT10 mit dem UBL-UBA Protein NEDD8 Ultimate Buster 1-Long (NUB1L). Ziel dieser Doktorarbeit war es, einen tieferen Einblick in den Prozess des FAT10- vermittelten Proteinabbaus zu erlangen. Des weiteren wurde ein monoklonaler Antikörper gegen humanes FAT10 hergestellt und es konnte gezeigt werden, dass FAT10 für die korrekte Herrunterregulation der CD8 + T Zell-vermittelten anti- viralen Immunantwort benötigt wird. Bereits im Vorfeld konnte gezeigt werden, dass NUB1L, welches über einen Yeast Two-Hybrid Screen als nicht-kovalenter Interaktionspartner von FAT10 identifiziert wurde, durch seine Co-Expression zusammen mit FAT10 dessen Ab- bau beschleunigen konnte. Die vorliegende Arbeit untersucht die molekularen Faktoren dieser Beschleunigung und deckt eine doppelte Rolle für NUB1L sowohl 6
Page 1 and 2: Role of the ubiquitin-like modifier
Page 3 and 4: Acknowledgements . . . . . . . . .
Page 5: Summary / Zusammenfassung English F
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
Page 55 and 56: 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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