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

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Chapter 2 Figure 15: The degradation of FAT10-DHFR by the 26S proteasome in vitro is independent of ubiquitylation but relies on NUB1L. The Y-axis always represents percent of acid soluble counts per hour. The in vitro reactions were performed with the components indicated above each panel. Inhib. refers to degradation obtained in the presence of the specific proteasome inhibitor epoxomycine. (A) Antizyme (AZ) dependent degradation of ornithin decarboxylase (ODC) as positive control for 26S proteasome activity. (B) HA-Ubiquitin-DHFR (Ub-DHFR) is not significanly degraded by the purified 26S proteasome. (C) NUB1L-dependent degradation of HA-FAT10-DHFR by purified 26S proteasome. A small and a normal sized (+) sign designate degradation in the presence of low (2ng) and high (20ng) amounts of NUB1L, respectively. 67
Chapter 2 evaluation of the NUB1L bands obtained by three dilutions of lysates on graded semi-quantitative Western blots (Fig. 16A) revealed that the residual expression of NUB1L protein in the shRNA transfectants was 2% (G6), 8.5% (F9), and 4% (H10), respectively. As NUB1L is the dominant isoform in HeLa cells, no NUB1 was detectable even in wild-type cells. Figure 16: Efficient knock down of NUB1L in stable HeLa transfectants expressing two NUB1L shRNAs. Shown are Western blots of wild-type HeLa-cells (WT) and 3 different stable transfectants expressing two NUB1L specific shRNAs (clones F9, G6 and H10). 10, 5 and 1µl of cell lysate (indicated above the panels) were loaded for each cell type. (A,C Western blots probed with two different NUB1/NUB1L peptide specific antibodies (A, van der Spuy et al., 2003); (B, Biomol). The arrowheads on the left show the position of NUB1L.(B, D Loading control for panels (A) and (C) with anti-HSP90 antibody. Next, we transiently transfected the three NUB1L knock-down clones as well as wild-type HeLa cells with an expression contruct for HA-FAT10-DHFR (Hipp et al., 2005). Subsequent to transfection, the degradation of HA-FAT10-DHFR was monitored in pulse-chase experiments. As shown in figure 17, the degradation of HA-FAT10-DHFR was markedly slowed down in the three NUB1L knock- down clones compared to wild-type HeLa cells, and the reduction in HA-FAT10- DHFR degradation correlated with the extent of NUB1L deficiency. This result strongly suggests that in intact cells, NUB1L is required for the degradation of FAT10-linked proteins by the proteasome. 68
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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
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 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: Chapter 2 Figure 14: Purified compo
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
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
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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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