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

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Chapter 3 in figure 19B and treated with proteasome inhibitor. In these experiments, we were able to identify the first deacetylase (CAT1) domain of HDAC6 as a second domain responsible for interacting with FAT10. Interestingly, this is not the domain which exhibits α-tubulin deacetylase activity (Haggarty et al., 2003). Both the isolated BUZ as well as the CAT1 domain were able to associate with FAT10 after proteasome inhibition, but not as strongly as the full length protein. However, the longer proteins which lack only the N-terminal (∆N) or C-terminal (∆BUZ) domain were able to precipitate amounts of FAT10 which were comparable to those pulled down by wild-type HDAC6. The 1-503 mutant showed weak interaction with FAT10 only in some of the experiments performed, while the 1-840 mutant was never able to bind FAT10, which might be explained by incor- rect folding or the formation of multimers which could result in blockage of the FAT10 binding site. To confirm the results obtained in cell lines in an in vitro setting, we performed a GST-pulldown assay using recombinant GST-FAT10 and in vitro transcribed and translated [ 35 S]-methionine labeled HDAC6. As shown in figure 20A, only GST-FAT10, but not GST alone, was able to pull down the CAT1 and the BUZ domain as well as full length HDAC6. Both of the isolated domains showed robust interaction with FAT10, while the isolated BUZ domain only interacted weakly in the co-immunoprecipitation experiments. In vitro as well as in intact cells, the full length protein displayed the highest affinity for FAT10. Taken together, these results demonstrate that both the BUZ as well as the CAT1 domain are sufficient to bind FAT10 on their own, but do so much more efficiently in the context of the full length protein. Binding of FAT10 to HDAC6 is not mediated through its C-terminal diglycine motif Previous studies have suggested the interaction between HDAC6 and ubiquitin to be largely mediated by its free C-terminal diglycine (Pai et al., 2007; Reyes-Turcu et al., 2006), therefore we decided to investigate whether the C-terminal diglycine motif of FAT10 was required for its interaction with HDAC6. As demonstrated in figure 20C, both wild-type FAT10 as well as a mutant lacking the C-terminal diglycine were able to pull down comparable amounts of radiolabeled HDAC6. Further evidence that its C-terminal diglycine is not required comes from experiments in which a linear fusion of FAT10 to GFP was transiently transfected into HEK293T cells. In this fusion protein, the two C-terminal glycines of FAT10 were exchanged for alanine and valine (AV), yet it was still able to localize to aggresomes (Fig. 23A) and could still be coprecipitated with HDAC6 (Fig. 26A). 83
Chapter 3 Figure 19: Both the BUZ domain and the first catalytic domain (CAT1) of HDAC6 are able to bind FAT10. (A) HEK293T cells, transfected with HA-FAT10 and the indicated FLAG-HDAC6 mutants, were treated with 5µM MG132 or DMSO as a negative control for 6 hours. Cell lysates were subjected to anti-FLAG immunoprecipitation (IP) and analysis by Western blot (WB). Asterisks denote the position of antibody light chains. (B) Schematic representation of the mutants used in A. CAT1 = first catalytic domain, CAT2 = second catalytic doman, BUZ = ubiquitin binding zinc finger. 84
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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
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
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: Chapter 3 Figure 18: HDAC6 interact
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 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
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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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