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

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Chapter 1 plex, Rpt6, demonstrates successful immunoprecipitation of the 26S proteasome in all cases (Fig. 13E and F). The ability to interact with the 26S proteasome was shown for FAT10-N-GFP and FAT10-C-GFP in the same manner, using GFP alone as negative and FAT10-GFP as positive control (Fig. 13B and D). The speci- ficity of the binding was again demonstrated by the lack of co-immunoprecipitation of GFP alone, despite successful immunoprecipitation of the 26S complex (Fig. 13F). To investigate whether the interaction between FAT10 and the proteasome is di- rect, an in vitro pull-down assay was performed where recombinant GST-FAT10 was incubated together with purified 26S proteasome. Only GST-FAT10, but not GST alone, was able to pull down the proteasome, as determined by a western blot against the 20S subunit iota (α1, Fig. 13G) although the same amount of proteasomes was available for interaction as demonstrated by western blotting (Fig. 13H). Discussion The role of proteins which contain both a UBL domain and one or more UBA domains for the recruitment of polyubiquitylated substrates for degradation by the proteasome is currently a subject of intensive investigations (Miller and Gordon, 2005). While it was previously believed that polyubiquitylation is a sufficient la- bel for docking to the 26S proteasome via the subunits Rpn10 (van Nocker et al., 1996) or Rpt5 (Lam et al., 2002), it is now becoming clear that UBL-UBA proteins, which bind to proteasomes via their UBL domain, can be required to target a subset of ubiquitylated substrates to the proteasome thus introducing a further layer of regulation. Here we investigated the molecular interactions required for a novel proteasomal targeting system consisting of the ubiquitin-like protein FAT10 and the UBL-UBA protein NUB1L. In table 1 we summarize the data we obtained while dissecting the different domains of NUB1L and FAT10 for interaction and accelerated degradation. When we identified NUB1L as a non-covalent interaction partner of FAT10 that contains three bona fide UBA domains, it was an obvious assumption that these are required for the interaction with FAT10. Since the alternative splice vari- ant NUB1 can be viewed as a ‘naturally occurring’ deletion mutant of NUB1L, it appeared reasonable to delete the UBA domains singly, in pairs, and altogether. We found in GST-pulldown experiments (Fig. 6B) and co-immunoprecipitation 51
Chapter 1 Figure 13: Co-immunoprecipitation of NUB1L, NUB1L∆UBA1-3, and FAT10, but not of NUB1L∆UBL with the 26S proteasome. (A) HEK293T cells were transfected with empty vector (mock), HA-NUB1L together with GFP (vector: pBI), HA-FAT10 together with GFP (vector: pBI), HA-NUB1L together with FAT10 (vector: pBI), HA-NUB1L (vector: pcDNA3.1), HA-NUB1L∆UBA1-3 (vector: pcDNA3.1) and HA-NUB1L∆UBL (vector: pcDNA3.1). The cells were then lysed and about 2% of the lysate was analyzed by SDS-PAGE and anti-HA Western blotting (WB). (B) HEK293T cells were transfected with GFP, HA-FAT10-GFP fusion protein, HA-FAT10-N-terminal UBL domain-GFP fusion protein (HA-Fat10-N-GFP), HA-FAT10-C-terminal UBL domain-GFP fusion protein (HA-FAT10-C- GFP), HA-NUB1L together with GFP (vector: pBI) and HA-FAT10 together with GFP (vector: pBI). The cells were lysed and about 2% of the lysate was analyzed by SDS-PAGE and anti-GFP Western blotting (WB). The positions of molecular weight markers are shown on the right side of the gel. (C, D) The lysates shown in A and B were subjected to immunoprecipitation with an antibody against the 20S proteasome subunit β7 (MCP444, anti-HN3) under conditions preserving the 26S proteasome. The bound proteins were analyzed by WB with an anti-HA antibody in C, or with an anti-GFP antibody in D. The lane occupation in C is the same as in A, the lane occupation in D is the same as in B. (E, F) To demonstrate the successful immunoprecipitation of the 26S proteasome, the samples shown in C and D were also analyzed by WB with an antibody recognising the 19S regulator subunit Rpt6. The lane occupation in E is the same as in A; the lane occupation in F is the same as in B. (G) GST-pull down experiment. GST (lanes 1 and 2) or GST-FAT10 (lanes 3 and 4) were incubated with purified 26S proteasome in the presence or absence of recombinant His 6-NUB1L and analyzed by WB with an antibody recognizing the 20S proteasome subunit iota (α1). (H) Supernatants of the GST-pull down shown in G were analyzed on a Western blot for iota content. The data were confirmed in three independent experiments. 52
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 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: Chapter 1 Figure 12: Accelerated de
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 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
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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
Page 113 and 114:
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
Page 123 and 124:
Discussion monomeric ubiquitin as w
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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
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Abbreviations aa amino acid AAA ATP
Page 145 and 146:
Amino Acids Alanine Ala A Arginine
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