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

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Chapter 3 Figure 22: FAT10 localizes to aggresomes under proteasome inhibition. (A) HEK293T cells transiently transfected with HA-FAT10 and FLAG-HDAC6 were treated with 5µM MG132 or DMSO as a negative control for 6 hours and immunostained with antibodies for HA (green), FLAG (red) and DAPI (blue; a-h). HEK293T cells transfected with HA-FAT10 were treated with 5µM MG132 or DMSO for 6 hours and immunostained with anti-HA (green), DAPI (blue) and anti-γ-tubulin (red; i-p), anti-Ubiquitin (red; q-t) or anti-20S proteasome (red; u-x). (B) HEK293T cells treated with TNF-α and IFN-γ for 16 hours were incubated in the presence of 5µM MG132 or DMSO for an additional 6 hours and immunostained with antibodies against endogenous FAT10 (green), Ubiquitin (red) and DAPI (blue). Scale bars = 5µm. Instead of localizing to one prominent juxtanuclear aggresome upon proteasome inhibition (Fig. 24A, d-f), FAT10 accumulated under nocodazole treatment in several microaggregates which were evenly dispersed throughout the cytoplasm (Fig. 24A, g-i). Since catalytic activity of HDAC6 is required for the transport of poly- ubiquitylated proteins to the aggresome (Kawaguchi et al., 2003), but is dispens- able for the interaction with FAT10 (Fig. 24 and 28), we set out to investigate the effect of the HDAC6 specific inhibitor tubacin (Haggarty et al., 2003) on the formation of FAT10 containing aggresomes. Unfortunately though, we were not able to draw many conclusions regarding the formation of aggresomes from this exper- iment, since the combination of tubacin and MG132 lead to profound cell death in FAT10 transfected cells already a few hours after treatment. Up until that point, however, the formation of aggresomes appeared to be comparable between cells treated with tubacin or the inactive control compound niltubacin (data not shown). Thus it is clear that the transport of FAT10 to the aggresome requires a functional microtubule network, but the question whether it also requires the deacetylase function of HDAC6 remains unsolved. 91
Chapter 3 Figure 23: The model conjugate FAT10-GFP localizes to aggresomes under proteasome inhibition. (A) HEK293T cells transfected with FLAG-HDAC6, GFP or FAT10-GFP were treated with 5µM MG132 or DMSO for 6 hours and immunostained with anti-GFP antibody (green), anti-FLAG antibody (red) and DAPI (blue). Scale bars = 5µm. (B) Quantification of percentage of cells with GFP containing aggresomes, n ≥ 100. Error bars represent the standard error of the mean (s.e.m.), statistical analysis was carried out using Student's t test. 92
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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
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Chapter 1 The UBA domains of NUB1L
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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 and 88: Chapter 3 of HDAC6 was required for
Page 89 and 90: Chapter 3 precipitates with HDAC6 (
Page 91: 90 Chapter 3
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,
Page 139 and 140: References P. Schreiner, X. Chen, K
Page 141 and 142: 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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