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

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Chapter 3 Figure 27: Neither acetylation of FAT10 nor catalytic activity of the CAT1 domain are required for its interaction with FAT10. (A) HEK293T cells were transfected with either catalytically active (wt) or inactive (mut) FLAG-CAT1 together with either wild-type or lysine-less (K0) HA-FAT10, followed by treatment with 5µM MG132 or DMSO for 6 h. Cell lysated were subjected to anti-FLAG immunoprecipitation and subsequent analysis by Western blot (WB). (B) GST-pulldown of an [ 35 S]-methionine labeled in vitro transcibed and translated active (wt) or inactive (mut) CAT1 domain by recombinant GST-FAT10. ished FAT10 binding (Fig. 21A-C), as (2) the mutation of all lysine residues of FAT10 did not alter its association with HDAC6 (Fig. 21D), and as (3) acetylated FAT10 did not preferentially associate with HDAC6 (Fig. 21E). Much more revealing were our experiments investigating the hypothesis that HDAC6 might also shuttle FAT10 to the aggresome in situations of misfolded protein stress or proteasome inhibition (Johnston et al., 1998; Kawaguchi et al., 2003). Indeed, proteasome inhibition lead to the accumulation of FAT10 or FAT10- linked GFP in a single, large juxtanuclear inclusion body. Compelling evidence has been collected that this inclusion body is an aggresome: firstly, FAT10 colo- calized in this structure with typical constituent proteins of aggresomes such as HDAC6, γ-tubulin, polyubiquitin, and the proteasome (Fig. 21). Secondly, FAT10 localized to this inclusion body only when the proteasome was inhibited, and did so with a kinetic similar to the one reported for aggresome formation (Garcia-Mata et al., 1999, Fig. 22 and data not shown). Thirdly, the presence of an intact microtubule network, which is essential for the formation of aggresomes, was also required as treatment of cells with the microtubule depolymerizing agent nocodazole in addition to proteasome inhibition resulted in the formation of mul- tiple FAT10 containing microaggregates in the cytosol (Fig. 24A). Evidence that the transport of FAT10 to aggresomes is not merely an artifact of overexpression comes from experiments in which endogenous FAT10, which was physiologically induced with IFN-γ and TNF-α, displayed a localization similar to that of the transiently transfected protein (Fig. 22B). In addition, co-immunoprecipitation 97
Chapter 3 also revealed endogenous, cytokine-induced FAT10 to be associated with HDAC6 (Fig. 18B). The question whether transport of FAT10 to the aggresome is solely dependent on HDAC6 was adressed in cells derived from HDAC6-deficient mice. A previous study with cells stably expressing an shRNA directed against HDAC6 (Kawaguchi et al., 2003) revealed HDAC6 to be required for the proper formation of polyubiquitin-containing aggresomes, as HDAC6 knock-down cells contained fewer and smaller aggresomes as compared to wild-type cells. However, it was impossible to determine whether the residual formation of aggresomes was due to an incomplete knock-down of HDAC6 or a functional redundancy. Our experiments in cells lacking the hdac6 gene now point toward the latter, as these cells display a similar phenotype (Fig. 24B, 25B and C). We were able to demonstrate the transport of FAT10 and polyubiquitin to the aggresome to be equally dependent on HDAC6; both FAT10- as well as polyubiquitin-containing aggresomes were significantly reduced in size in HDAC6-deficient compared to wild-type cells (Fig. 25C and D). Figure 28: Inhibition of catalytic activity has no effect on the interaction between HDAC6 and FAT10 in vitro . GST-pulldown of [ 35 S]-methionine labeled in vitro transrcibed and translated HDAC6 with recombinant GST-FAT10 in the presence of 20µM TSA or 160µM tubacin. The role of tubulin deacetylation in the transport of FAT10 to the aggresome however remains unclear. We attempted to investigate whether the deacetylase activity of HDAC6 was required for the transfer of FAT10 into aggresomes by using the HDAC6 specific inhibitor tubacin. Unfortunately, cells treated with proteasome inhibitor and tubacin at the same time did not survive long enough to investigate aggresome formation. We only can say for certain that the catalytic activity of HDAC6 is dispensable for its interaction with FAT10, as both inhibition as well as mutation of the active sites failed to abolish this interaction (Figs. 21 and 28). In fact, the association of HDAC6 with FAT10 was even increased after treatment of cells with the broad-spectrum deacetylase inhibitor TSA (Fig. 21B), which might be attributed to an increase in α-tubulin acetylation as well as non-specific effects 98
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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
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Chapter 1 no correlation between th
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Chapter 1 covered proteins were sep
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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 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: Chapter 3 Figure 26: Both ubiquitin
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
Page 143 and 144: Abbreviations aa amino acid AAA ATP
Page 145 and 146: Amino Acids Alanine Ala A Arginine
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