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

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Chapter 3 of TSA on other targets, especially as treatment of cells with tubacin has a much less pronounced effect (Fig. 21C). The question arises why the interaction between HDAC6 and FAT10 can only be observed under proteasome inhibition. Boyault et al. have proposed a model in which HDAC6 senses an overload of polyubiquitylated proteins through the binding of polyubiquitin to its BUZ domain. This then results in the release of heat-shock factor 1 and HDAC6 from a complex also containing p97/VCP and HSP90 (Boyault et al., 2007). An alternative model (Pai et al., 2007) suggests that it is not the accumulation of polyubiquitylated proteins but rather the lack of unconjugated ubiquitin resulting from proteasome inhibition that facilitates the interaction of HDAC6 and polyubiquitin. Owing to a much higher affinity of the BUZ domain for the C-terminus of ubiquitin rather than for polyubiquitin- conjugates (Reyes-Turcu et al., 2006), the majority of HDAC6 would normally be bound to free ubiquitin. Under conditions of proteasome impairment, the decline in the level of monomeric ubiquitin releases HDAC6. In both models, HDACD6 would then be free to interact with polyubiquitin - and also FAT10-conjugates. Figure 29: FAT10 is acetylated but does not appear to be a substrate of HDAC6mediated deacetylation. (A) HEK293T cells transfected with HA-FAT10 were treated with 5µM MG132, 5µM TSA or mock treated for 6 h before lysis, immunoprecipitation (IP) with anti-FAT10 or control serum and subsequent analysis by Western blot (WB) with an acetyl-Lysine antibody (AcK). (B) HEK293T cells transfected with either HA- FAT10, FLAG-HDAC6 or empty vector were treated for 6 h with 5µM MG132 or DMSO before lysis, anti-FAT10 immunoprecipitation (IP) and analysis by Western blot (WB). Asterisks denote the position of antibody light chains, arrows the position of HA-FAT10 on the anti-AcK Western blots. It is interesting that FAT10, which can target proteins for proteasomal degradation independently of the ubiquitin system, uses the same rescue strategy when the proteasome is overwhelmed. This implies that the rapid removal of FAT10 99
Chapter 3 and/or its conjugates is essential for the cell, for example to protect it from the deleterious effects caused by an excess of free FAT10, which is suggested by the finding that the ectopic expression of FAT10 induces apoptosis in several cell types (Raasi et al., 2001; Ross et al., 2006). The formation of ubiquitin-containing aggregates is a hallmark of many neurode- generative diseases like Huntington’s or Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is notable that NUB1L, another non-covalent interaction partner of FAT10 (Hipp et al., 2004), accumulates in Lewy bodies of PD and DLB (Tanji et al., 2007) and localizes to aggresomes under proteasome inhibition (data not shown). It will hence be interesting to investigate whether FAT10 is also a component of Lewy bodies and may even be responsible for targeting NUB1L to these sites. Since the basal expression of FAT10 in the brain is extremely low (Canaan et al., 2006), an involvement of FAT10 would rely on a strong induc- tion of fat10 expression with TNF-α and IFN-γ which is probably not prevalent in these chronic neurological disorders. Recently, FAT10 was shown to be highly upregulated in hepatocellular carcinoma (Lee et al., 2003), and in a drug induced mouse model of hepatocellular carcinoma FAT10 localized to Mallory-Denk bodies, which are a special form of aggresomes observed in a variety of liver diseases (Oliva et al., 2008; Zatloukal et al., 2007). On the other hand, the localization of the fat10 gene in the major histocompatibility class I locus and the fact that it is inducible by proinflammatory cytokines point toward a function in the immune response. It remains subject to investigation whether FAT10 is perhaps involved in the formation of DALIS – which are transient aggresome like structures that function as storage compartments of antigenic proteins during the maturation of dendritic cells (Lelouard et al., 2002). In addition, as autophagy has been im- plicated in the innate as well as the adaptive immune response to intracellular pathogens, it is tempting to speculate whether FAT10 might be involved in those processes as well. Materials and Methods Tissue culture and transfection. Spontaneously immortalized HDAC6 +/+ and HDAC6 -/- MEFs were generously provided by P. Matthias (Zhang et al., 2008b). MEFs and HEK293T cells were cultivated in DMEM supplemented with 10% FCS and 100 µg/ml penicillin/streptomycin (all from Invitrogen). HEK293T cells were transiently transfected using FuGENE 6 (Roche) according to the manufac- turer’s instructions. MEFs were transiently transfected using the Amaxa MEF2 100
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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
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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 and 98: Chapter 3 Figure 26: Both ubiquitin
Page 99: Chapter 3 also revealed endogenous,
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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