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

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Abstract Chapter 2 FAT10 is a ubiquitin-like protein which is encoded in the major histocompatibil- ity complex class I locus and is synergistically inducible with interferon-gamma and tumor necrosis factor-alpha. It encompasses two ubiquitin-like domains connected by a short linker, and bears a free diglycine-motif at its C-terminus through which it forms covalent conjugates with its target proteins. Previously, we have shown that both monomeric FAT10 as well as its conjugates are rapidly degraded by the proteasome in intact cells and their degradation was further accelerated by expression of the UBL-UBA domain protein NEDD8 ultimate buster 1-long (NUB1L). This degradation was independent of a ubiquitin acti- vating enzyme and did not rely on the lysine residues of FAT10, suggesting that poly-ubiquitylation was not involved. Here we show that the 26S proteasome readily degrades the model substrate FAT10-dihydrofolate reductase (DHFR) but not ubiquitin-DHFR in vitro, demonstrating conclusively that FAT10-mediated degradation does not rely on poly-ubiquitylation. Interestingly, the purified 26S proteasome could only degrade FAT10-DHFR when NUB1L was present. Knock- down of NUB1L attenuated the degradation of FAT10-DHFR, suggesting that NUB1L modulates the degradation rate of FAT10-linked proteins in cells. In conclusion, our data establish FAT10 as a ubiquitin-independent but NUB1L- dependent targeting signal for proteasomal degradation. 61
Introduction Chapter 2 The proteasome is responsible for the turnover of the majority of intracellular proteins in eukaryotes and is essential for the degradation of misfolded proteins and the destruction of short-lived regulatory proteins. Most proteins are degraded by the 26S proteasome, a 2.4M Da multi-subunit complex consisting of the 20S proteasome core particle capped by one or two 19S regulatory particles (Hanna and Finley, 2007). The 20S proteasome is a cylindrical stack of four 7-membered rings around a central pore, where subunits α1-α7 make up the outer and subunits β1- β7 the inner two rings. Six of those subunits – two copies of β1, β2 and β5 – are catalytically active, and their active sites point toward the central lumen of the core particle. The 19S regulator, also known as PA700, can be further subdivided into the proximal “base”, which abuts to the outer α-rings of the 20S proteasome, and the distal “lid”. The “base” consists of six AAA-family ATPases (Rpt1-Rpt6) and four non-ATPase subunits (Rpn1, 2, 10 and 13). The ATPases form a hex- americ ring, which mediates ATP-dependent opening of the pores formed by the α-subunit rings and unfolding of substrate proteins. The remainder of the approx- imately 20 PA700 subunits make up the “lid”; and while some of them could be shown to display deubiquitylating activity, most of their functions are unknown. Proteins are targeted for proteasomal degradation by the covalent attachment of a chain of ubiquitin molecules by way of a multi-enzyme cascade. Ubiquitin first gets activated by an E1 enzyme, then it is transferred to an E2 enzyme and finally, through the help of an E3 enzyme, becomes conjugated to an acceptor lysine of a target protein. After attachment of a single ubiquitin molecule, ad- ditional ubiquitins can become conjugated to each of its seven lysine residues to form a polyubiquitin chain. Polyubiquitin chains linked through K48 are the principal targeting signal, with the attachment of a tetraubiquitin chain serv- ing as the minimal signal required for proteasomal degradation (Thrower et al., 2000). Substrate recognition of the proteasome is mediated by four subunits of the 19S regulator; S5a/Rpn10 and S6’/Rpt5, which directly recruit K48-linked polyubiquitin chains (Deveraux et al., 1994; Lam et al., 2002) as well as S1/Rpn2 and S2/Rpn1, which indirectly recognize substrates by binding to linker proteins such as Rad23/hHR23 and Dsk2/hPLIC (Elsasser et al., 2002; Saeki et al., 2002b; Walters et al., 2002). Aside from this canonical mode of proteasomal degradation, a few alternative routes into the proteasome exist. The most prominent and well-studied case is that of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the 62
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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
Page 11 and 12: The Ubiquitin-Proteasome-System The
Page 13 and 14: Introduction Figure 1: The 26S prot
Page 15 and 16: Introduction are the major source o
Page 17 and 18: 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: Chapter 2 Degradation of FAT10 by t
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 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
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Introduction Chapter 5 Antibodies a
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Chapter 5 binant human and mouse GS
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Chapter 5 Figure 34: The antibodies
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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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