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

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Chapter 3 Figure 21: Neither acetylation of FAT10, nor catalytic activity of HDAC6 are required for interaction between the two proteins. (A) HEK293T cells transfected with HA-FAT10 and either wild-type (wt) FLAG-HDAC6 or a catalytically dead mutant (mut) were treated with 5µM MG132 or DMSO as a negative control for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by Western blot (WB). (B) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5µM MG132, 1µM nocodazole, 5µM of the HDAC inhibitor trichostatin A (TSA) or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by Western blot (WB). (C) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5µM MG132, 20µM tubacin or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by Western blot (WB). (D) HEK293T cells, transfected with FLAG-HDAC6 and either wild-type (wt) or a lysine-less HA-FAT10 mutant (K0), were treated with 5µM MG132 or DMSO for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by Western blot (WB). (E) HEK293T cells transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5µM MG132, 5µM TSA or mock treated for 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by Western blot (WB) with an acetyl-Lysine antibody (AcK). An asterisk denotes the position of the antibody light chain, an arrow the position of HA-FAT10 on the anti-AcK Western blot. 87
Chapter 3 precipitates with HDAC6 (Fig. 21E). Moreover, a mutant of FAT10 in which all lysines were mutated to arginine (Hipp et al., 2005) – thus preventing it from being ubiquitylated or acetylated – was still able to interact with HDAC6 as well as, if not better than, wild-type FAT10 (Fig. 21D). Still, the ability of catalytically dead or inhibited HDAC6 to interact with FAT10 might solely be mediated through the C-terminal BUZ domain. To exclude this possibility, we performed co-immunoprecipitation experiments with an isolated, catalytically dead CAT1 domain. As can be seen in figure 27A, the mutated CAT1 domain was still able to pull down FAT10. In addition, the isolated CAT1 domain was still capable of interacting with the lysine-less mutant of FAT10. Figure 27B demonstrates that recombinant GST-FAT10 was able to pull down similar amounts of the wild-type and mutated in vitro transcribed and translated [ 35 S]-methionine labeled CAT1 domains. Together, these findings suggest that, while a small portion of the intra- cellular FAT10 is acetylated, acetylation is not required for its binding to HDAC6 nor is FAT10 a substrate of HDAC6 deacetylase activity. FAT10 and FAT10-GFP localize to the aggresome after proteasome inhibition As FAT10 interacts with HDAC6 after proteasome inhibition, and as HDAC6 is involved in the transport of polyubiquitylated proteins to the aggresome upon proteasome impairment (Kawaguchi et al., 2003), we investigated whether HDAC6 may likewise transport FAT10 to the aggresome. To test this hypothesis, we investigated whether treatment of transiently HA-FAT10-tranfected HEK293T cells with proteasome inhibitor had an influence on the subcellular localization of FAT10 as analyzed by confocal immunofluorescence microscopy. In untreated cells, FAT10 was evenly distributed throughout the cytoplasm and showed vary- ing degrees of localization to the nucleus (Fig. 22A, a and i). HDAC6, on the other hand, was completely excluded from the nucleus and showed only cytoplas- matic localization (Fig. 22A, b). As reported by Kawaguchi et al., proteasome inhibition induced relocalization of HDAC6 to a single prominent juxtanuclear structure – and indeed also caused FAT10 to localize to the very same structure (Fig. 22A, e-h). To determine the identity of the observed inclusion bodies, we analyzed them for the presence of known aggresome markers other than HDAC6. We never observed formation of more than one of these inclusion bodies per cell, and combined with the observation that they colocalizd with γ-tubulin (Fig. 22A, m-p), a component of centromeres, this leads us to infer that they form at the microtubule organizing center. In addition, we observed a characteristic distor- tion and enlargement of the centromeres upon proteasome inhibition (compare Fig. 22A, j and n), which is another hallmark of aggresome formation (Johnston 88
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Role of the ubiquitin-like modifier
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Acknowledgements . . . . . . . . .
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Summary / Zusammenfassung English F
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Summary In conclusion, these result
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Summary nicht von der katalytischen
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The Ubiquitin-Proteasome-System The
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Introduction Figure 1: The 26S prot
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Introduction are the major source o
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Introduction majority of ubiquitin-
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Introduction example contained in t
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Introduction two modifiers apart fr
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Introduction Figure 4: Comparison o
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Introduction dependent apoptosis in
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Introduction Interestingly, a recen
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The Autophagy-Lysosome System Intro
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Introduction Figure 5: Molecular ma
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Introduction The transport of misfo
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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
Page 57 and 58: Chapter 1 Rad23 (Verma et al., 2004
Page 59 and 60: Chapter 1 FAT10-GFP expressing vect
Page 61 and 62: Chapter 2 Degradation of FAT10 by t
Page 63 and 64: Introduction Chapter 2 The proteaso
Page 65 and 66: Chapter 2 From our previous data it
Page 67 and 68: Chapter 2 Figure 14: Purified compo
Page 69 and 70: Chapter 2 evaluation of the NUB1L b
Page 71 and 72: Chapter 2 teins which contain intri
Page 73 and 74: Chapter 2 Combined with our studies
Page 75 and 76: Chapter 2 Recombinant expression of
Page 77 and 78: Chapter 2 et al., 2003), both at a
Page 79 and 80: Abstract Chapter 3 During misfolded
Page 81 and 82: Chapter 3 tion between the UPS and
Page 83 and 84: Chapter 3 Figure 18: HDAC6 interact
Page 85 and 86: Chapter 3 Figure 19: Both the BUZ d
Page 87: Chapter 3 of HDAC6 was required for
Page 91 and 92: 90 Chapter 3
Page 93 and 94: Chapter 3 Figure 23: The model conj
Page 95 and 96: Chapter 3 HDAC6 is required for the
Page 97 and 98: Chapter 3 Figure 26: Both ubiquitin
Page 99 and 100: Chapter 3 also revealed endogenous,
Page 101 and 102: Chapter 3 and/or its conjugates is
Page 103 and 104: Chapter 3 (Hipp et al., 2005), pEGF
Page 105 and 106: Chapter 4 FAT10-deficient mice disp
Page 107 and 108: Chapter 4 Once the infection is cle
Page 109 and 110: Chapter 4 As both wild-type as well
Page 111 and 112: Chapter 4 Peptides. The synthetic p
Page 113 and 114: Introduction Chapter 5 Antibodies a
Page 115 and 116: Chapter 5 binant human and mouse GS
Page 117 and 118: Chapter 5 Figure 34: The antibodies
Page 119 and 120: Chapter 5 Figure 36: The antibody s
Page 121 and 122: Discussion 30 years ago, ubiquitin
Page 123 and 124: Discussion monomeric ubiquitin as w
Page 125 and 126: Discussion Alternatively, FAT10 cou
Page 127 and 128: References D. T. Akey, X. Zhu, M. D
Page 129 and 130: References Y.-H. Chiu, Q. Sun, and
Page 131 and 132: References S. Gunawardena, L.-S. He
Page 133 and 134: References J. A. Johnston, M. E. Il
Page 135 and 136: References F. Lévy, N. Johnsson, T
Page 137 and 138: 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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