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

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Introduction A distinct subgroup consists of ubiquitin domain proteins which are character- ized by the possession of one or more ubiquitin-associated (UBA) domains and are thought to function as soluble ubiquitin receptors and facilitators of proteasomal degradation. Members of this family of UBL-UBA domain proteins in- clude hHR23/Rad23 (Chen and Madura, 2002), hPLIC/ubiquilin/Dsk2 (Funakoshi et al., 2002), NUB1 (Kito et al., 2001), and Ddi1/Vsm1 (Bertolaet et al., 2001), which is the only member of this family containing a putative aspartyl protease domain (Sirkis et al., 2006). Although there are conflicting data, a growing body of evidence now supports a model in which UBL-UBA proteins function as proteasome-substrate carriers by binding to the proteasome through their UBL domain and to polyubiquitylated cargo through their UBA domain(s). Most of the evidence comes from the study of Rad23, which was originally dis- covered as a component of nuclear excision repair, and Dsk2. Both proteins could be shown to interact via their UBL domain with the S5a (Hiyama et al., 1999; Walters et al., 2002) and S2 (Elsasser et al., 2002) subunits of the proteasome and with polyubiquitylated cargo through their UBA domains (Chen et al., 2001; Zhang et al., 2008a). Through genetic studies, which were mostly performed in yeast, it could be shown that deletion of either of these proteins resulted in the accumulation of high molecular weight ubiquitin conjugates and impaired degradation of a limited number of proteasomal substrates (Chen and Madura, 2002; Funakoshi et al., 2002; Rao and Sastry, 2002). It would be expected that the overexpression of UBL-UBA proteins, in turn, stimulates the degradation of these substrates, which could indeed be observed in some cases (Funakoshi et al., 2002; Verma et al., 2004b). However, it appears that the system is highly sen- sitive to pertubations in the level of UBL-UBA proteins, as their overexpression often leads to the stabilization, rather than accelerated degradation, of proteasomal substrates (Kleijnen et al., 2000; Ortolan et al., 2000). This effect can probably be attributed to the sequestration of polyubiquin chains by an excess of soluble UBA domain containing proteins, which effectively prevents their as- sociation with all sorts of ubiquitin-interacting proteins including not only proteasomal subunits (Raasi and Pickart, 2003), but also for example enzymes in- volved in polyubiquitin-chain assembly (Ortolan et al., 2000), or deubiquitylation (Hartmann-Petersen et al., 2003). As most manipulations concerning a single proteasome receptor have only mild effects on the bulk turnover of polyubiquitylated proteins, there appears to be a high level of redundancy in the delivery of polyubiquitylated substrates to the proteasome. Accordingly, only the combined deletion of the three proteasome receptors Rpn10, Rad23 and Dsk2 results in a severe (Saeki et al., 2002a) or lethal (Wilkinson et al., 2001) phenotype in yeast. 25
Introduction Interestingly, a recent study has uncovered a dual role of these ubiquitin-receptors in both substrate delivery and the stimulation of proteasomal activity (Verma et al., 2004a). Although Rad23 was sufficient to physically link a polyubiquitylated substrate to the proteasome, the presence of the VWA domain of Rpn10 – but not its UIM domain – was required to facilitate its degradation. As this requirement for facilitation was only observed for one of the substrates under investigation, it appears that despite of all redunancy, different substrates are preferrentially targeted to the proteasome via different mechanisms. NUB1 NUB1 was originally identified as a protein able to interact with and down- regulate cellular NEDD8 levels via proteasomal degradation (Kito et al., 2001; Kamitani et al., 2001) and was thus christened “NEDD8 ultimate buster 1”. It is a member of the family of UBL-UBA domain proteins and exists in the form of two splicing variants, a short version of 601 amino acids in length (NUB1), which contains two UBA domains, and a long version (termed NUB1long or NUB1L) containing an insertion of 14 amino acids, which generates a third UBA domain sandwiched inbetween the two existing ones (Tanaka et al., 2003). NUB1 has a somewhat unique status among the UBL-UBA proteins, as it is the only member of this family which contains UBA domains that are unable to associate with either polyubiquitin-chains or monoubiquitin (Raasi et al., 2005). The binding of NEDD8 was mapped to the second UBA domain (UBA2) as well as a short sequence near the C-terminus of NUB1, which includes the C-terminal half of a PEST domain. In addition, NUB1 was also reported to interact with the ubiquitin-precursor UbC1, however, the functional significance of this finding re- mains unknown (Tanaka et al., 2004). In addition, it is able to associate with the proteasomal subunit S5a/Rpn10 (Kamitani et al., 2001), and this interaction has surprisingly been reported to be mediated not through its UBL domain but rather through a short C-terminal region between amino acids 536 and 584 (Tanji et al., 2005). NUB1 is inducible with IFN-β as well as IFN-γ and its subcellular localization is mainly nuclear, owing to the presense of a nuclear localization sig- nal (NLS) between residues 414 and 431 (Kito et al., 2001). Even though it is cytokine-inducible, NUB1/NUB1L appears to be universally expressed in all tis- sue types, although the two isoforms differ in their level of mRNA expression (Tanaka et al., 2003). 26
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: Introduction dependent apoptosis in
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 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
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Chapter 2 et al., 2003), both at a
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Abstract Chapter 3 During misfolded
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Chapter 3 tion between the UPS and
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Chapter 3 Figure 18: HDAC6 interact
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Chapter 3 Figure 19: Both the BUZ d
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Chapter 3 of HDAC6 was required for
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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
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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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