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

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Abstract Chapter 1 Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA-domains and the 26S proteasome via their UBL-domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and con- tains one UBL- and three UBA-domains. We recently reported that NUB1L inter- acts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL-domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA-domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL-domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL-domains alone can interact directly with the 26S proteasome. We propose that NUB1L not only acts as a linker between the 26S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation. 37
Introduction Chapter 1 Ubiquitin, a small protein of 76 amino acids is one of the most conserved proteins known and has been found in all eukaryotic cells studied. It is essential for a variety of cellular processes, including degradation, cell-cycle regula- tion, DNA repair, stress response, embryogenesis, apoptosis, signal transduc- tion, and transmembrane- and vesicular transport. (Pickart, 2001; Hicke, 2001; Jesenberger and Jentsch, 2002; Lai, 2002; Ma and Hendershot, 2001; Koepp et al., 1999). Throughout the past years, a family of proteins containing structural motives related to ubiquitin has been described which can be grouped into the ubiquitin-like modifiers and the ubiquitin-domain proteins (Jentsch and Pyrowolakis, 2000). The ubiquitin-like modifiers have substantial sequence or structural homology to ubiquitin and form covalent conjugates with their target proteins. However, unlike ubiquitin, which can form large polymeric conjugates, usually only monomeric modifications are observed. As is the case for ubiquitin, a C-terminal diglycine motif is essential for conjugation of most modifiers to their target proteins (Yeh et al., 2000). Prominent members of this group include SUMO-1, which serves several functions including nuclear transport and bud- ding, (Matunis et al., 1996; Müller et al., 2001), NEDD8, which regulates SCF ubiquitin-ligases via cullin modification (Osaka et al., 2000), ISG15, which plays a role in innate immunity and in the response to alpha interferon, (Ritchie et al., 2004; Kim et al., 2005) and FAT10, which is inducible with gamma interferon (IFN)-γ and tumour necrosis factor alpha (TNF)-α (Raasi et al., 1999; Liu et al., 1999), and has been shown to cause apoptosis upon ectopic expression (Raasi et al., 2001). The ubiquitin-like protein FAT10 consists of two UBL domains in a head to tail formation, which are about 29% and 36% identical to ubiquitin, respectively. Sev- eral key features of ubiquitin, like the lysine residues 48, 63, and 29, which are required for polyubiquitin chain formation, are conserved in both UBL-domains (Fan et al., 1996). Its C-terminus bears a free diglycine motif, which is neces- sary for the conjugation to so far unidentified target proteins (Raasi et al., 2001). Like ubiquitin (Johnson et al., 1992), FAT10 causes rapid degradation of long- lived proteins when fused to the N-terminus (Hipp et al., 2005). However, unlike ubiquitin, which is recycled from the degraded target proteins, FAT10 is digested along with its substrates. FAT10 thus has a relatively short half life (Hipp et al., 2004), which decreases dramatically by coexpression of a member of the group 38
Page 1 and 2: Role of the ubiquitin-like modifier
Page 3 and 4: Acknowledgements . . . . . . . . .
Page 5 and 6: Summary / Zusammenfassung English F
Page 7 and 8: Summary In conclusion, these result
Page 9 and 10: 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: Chapter 1 The UBA domains of NUB1L
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 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 (
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90 Chapter 3
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Chapter 3 Figure 23: The model conj
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Chapter 3 HDAC6 is required for the
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Chapter 3 Figure 26: Both ubiquitin
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Chapter 3 also revealed endogenous,
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Chapter 3 and/or its conjugates is
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Chapter 3 (Hipp et al., 2005), pEGF
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Chapter 4 FAT10-deficient mice disp
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Chapter 4 Once the infection is cle
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Chapter 4 As both wild-type as well
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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
Page 137 and 138:
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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