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

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Chapter 3 of HDAC6. Combined with previous findings, these results suggest that the role of FAT10 is the rapid and inducible destruction of its target proteins via conju- gation and subsequent degradation. If the primary way of degradation by the proteasome fails, an alternative route is taken to ensure their removal by seques- tering them in the aggresome. Results FAT10 interacts with HDAC6 after proteasome inhibition A yeast two-hybrid screen using FAT10 as a bait against a human lymph node cDNA library (Hipp et al., 2004) identified HDAC6 as a novel interaction partner of FAT10. Sequencing of the plasmid recovered from the transformant revealed that the insert did not encode a full length copy of HDAC6 but rather an N-terminal truncation containing only the last 106 amino acids of the protein. This C-terminal part of HDAC6 encompasses little more than the zinc-finger domain (BUZ) which is responsible for the interaction with free ubiquitin and ubiquitin conjugates (Seigneurin-Berny et al., 2001; Hook et al., 2002). To confirm the interaction with FAT10 in vivo, we transfected HEK293T cells with FLAG-tagged HDAC6 as well as hemagglutinin (HA)-tagged FAT10 and treated them with proteasome inhibitor before performing co-immunoprecipitation experiments. In ac- cordance with the finding that HDAC6 only associated non-covalently with polyu- biquitylated proteins if the proteasome is impaired (Kawaguchi et al., 2003), we observed robust interaction of FAT10 with wild-type HDAC6 only after proteasome inhibition (Fig. 18A, lanes 1 and 2). As shown in figure 18B, transiently transfected HDAC6 was also capable of interacting with endogenous FAT10 after proteasome inhibition. To our surprise, deletion of the BUZ domain did not abolish the interaction between FAT10 and HDAC6 (Fig. 18A, lanes 3 and 4), sug- gesting that – while being able to bind FAT10 on its own as shown by the yeast two-hybrid screen – the BUZ domain is not the only domain of HDAC6 which is capable of binding to FAT10. HDAC6 has no influence on the rate of FAT10 degradation Since overexpression of the other interaction partner discovered in the yeast- two hybrid screen, the ubiquitin-domain protein NEDD8 ultimate buster-1 long (NUB1L), leads to a marked acceleration of FAT10 degradation (Hipp et al., 2004), and since HDAC6 is able to attenuate the proteasomal degradation of poly- 81
Chapter 3 Figure 18: HDAC6 interacts with FAT10 only under proteasome inhibition and has no influence on the degradation rate of FAT10. (A) HEK293T cells, transiently transfected with HA-FAT10 and either wild-type (wt) FLAG-HDAC6, a ∆BUZ mutant, or empty vector, were treated with 5µM of the proteasome inhibitor 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 were transiently transfected with FLAG-HDAC6 followed by treatment with 500 U/ml TNF-α and 200 U/ml IFN-γ for 16 hours and 5µM MG132 for another 6 hours before lysis, anti-FLAG immunoprecipitation (IP) and analysis by Western blot (WB). (C) HEK293T cells transfected with either HA-FAT10 alone (left panel), HA-FAT10 and FLAG-HDAC6 together (center panel) or FLAG-HDAC6 alone (right panel) were pulse labeled with [ 35 S]-methionine and chased for the indicated times. Cell lysates were subjected to anti-HA and anti-FLAG immunoprecipitation followed by SDS-PAGE and autoradiography. ubiquitylated misfolded proteins (Boyault et al., 2006), we decided to investigate whether overexpression of HDAC6 had an effect on the rate of FAT10 degradation. We performed pulse-chase assays with FAT10 in the presence or absence of HDAC6, however, as can be seen in 18C, coexpression of HDAC6 did not affect the degradation of FAT10. HDAC6 binds FAT10 via the zinc finger (BUZ) and the first catalytic domain (CAT1) To identify additional FAT10-binding domains of HDAC6, we performed co- immunoprecipitation experiments with truncation mutants of HDAC6. To this aim, cells were transfected with FAT10 and each of the HDAC6 mutants depicted 82
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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
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
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: Chapter 3 tion between the UPS and
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 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
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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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