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

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Chapter 2 Figure 17: Knock-down of NUB1L attenuates the degradation of HA-FAT10-DHFR. (A) Shown are autoradiograms of HA-FAT10-DHFR immunoprecipitations from wild-type HeLa cells (WT) and the NUB1L shRNA transfected clones G6, F9, und H10 described in figure 3. Cells were pulse labeled with [ 35 S]-methionine/cysteine for one hour and subsequently chased for the indicated time periods (in h). (B) Graphic representation of the phosphorimager-assisted quantitative evaluation of three different experiments as exemplified in panel (A); the extent of FAT10 degradation in percent is plotted versus the chase time. Shown are the means +/-SD for HeLa wild-type cells (diamonds), and the transfectants F9 (dots), H10 (triangles), and G6 (cubes). Discussion Ubiquitin has long lost its unique status as the sole mediator of proteasomal degradation. While other, non-proteolytic functions of ubiquitin have been dis- covered, several alternative routes into the proteasome have also been scouted. The ubiquitin-like modifier FAT10 was shown to be as potent as ubiquitin in targeting artificial fusion proteins for proteasomal degradation. The present study shows that FAT10-linked proteins are efficiently degraded by the 26S proteasome in vitro, but only in the presence of the UBL-UBA domain protein NUB1L. Degradation by the proteasome can be divided into two main categories: in- ducible destruction, which is chiefly mediated by attachment of a K48-linked polyubiquitin chain to the target protein, and degradation “by default” of pro- 69
Chapter 2 teins which contain intrinsic targeting signals (Asher et al., 2006). A diverse group of proteins has been described which can be characterized by the presence of one or more loosely folded domains and which can be degraded by the 20S proteasome without the need for prior ubiquitylation or ATP-dependent unfolding. Members of this group include IκBα (Alvarez-Castelao and Castaño, 2005), p53 (Asher et al., 2005), p21 Waf1/Cip1 (Touitou et al., 2001), α-synuclein (Tofaris et al., 2001), thymidylate synthase (Forsthoefel et al., 2004), and ODC (Asher et al., 2005). As many of these proteins function as part of a multimeric complex, and “default” degradation can only be observed when they are present in monomeric form, it has been suggested that assembly into a larger complex masks the un- structured regions – which are considered to function as the targeting signal – and thereby protects them from degradation. It is interesting to note that most, if not all of these proteins can also be actively targeted to the 26S proteasome by attachment of a polyubiquitin chain or – in the case of ODC – by interaction with AZ. The structure of FAT10 remains yet to be solved, however, the high sequence sim- ilarity to ubiquitin and ISG15 suggests that it is in all likelihood composed of two ubiquitin-fold domains (Narasimhan et al., 2005), which are anything but loosely folded. One can thus safely assume that FAT10 has evolved as a component of a specialized regulatory system responsible for targeting a subset of proteins for proteasomal degradation, and is not merely degraded due to an intrinsic instabil- ity. This notion is further supported by the finding that purified 20S proteasome – which was fully competent in the cleavage of short peptide substrates (data not shown) – was unable to degrade HA-FAT10-DHFR in vitro (Fig. 15C), and that C-terminal attachment of a FAT10-tag does not promote degradation (data not shown). The precise mechanism by which FAT10 is targeted to the proteasome has some- what remained a mystery. Discovery of the UBL-UBA domain protein NUB1L as a non-covalent interaction partner and an accelerator of FAT10 degradation (Hipp et al., 2004) suggested that it might interact with FAT10 through its C-terminal UBA domains and with the 19S regulator through its N-terminal UBL domain and thus physically link FAT10 to the proteasome. It was soon revealed, however, that while NUB1L did interact with FAT10 through all three of its UBA domains, only the UBL domain was required to accelerate its degradation, and that FAT10 was perfectly able to interact with the 26S proteasome on its own (Schmidtke et al., 2006). Recent studies investigating other members of this family, most notably Rad23/hHR23 and Dsk2/hPLIC, have revealed functional redundancy in 70
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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-
Page 19 and 20: Introduction example contained in t
Page 21 and 22: Introduction two modifiers apart fr
Page 23 and 24: Introduction Figure 4: Comparison o
Page 25 and 26: Introduction dependent apoptosis in
Page 27 and 28: Introduction Interestingly, a recen
Page 29 and 30: 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: Chapter 2 evaluation of the NUB1L b
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 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
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Discussion 30 years ago, ubiquitin
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Discussion monomeric ubiquitin as w
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Discussion Alternatively, FAT10 cou
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References D. T. Akey, X. Zhu, M. D
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References Y.-H. Chiu, Q. Sun, and
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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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