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

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Chapter 1 Figure 6: Domain structure of NUB1 and NUB1L, some deletion mutants, and their interaction with GST and GST-FAT10. (A) The positions of the domains of NUB1L are indicated by the number of the amino acids (aa). The ubiquitin-like (UBL) domains are shown in grey, the ubiquitin-associated (UBA) domains are shown in white. (B) Autoradiography of in vitro transcribed and translated NUB1L mutants after separation by SDS-PAGE. A 10% aliquot of each reaction was loaded to demonstrate the amount available for the GST pull-down. The positions of molecular weight markers (in kD) are indicated on the right. (C) Autoradiography of the NUB1L mutants shown in A after incubation with GST coupled GSH-sepharose, washing and separation by SDS-PAGE. The lane occupation is the same as in B. (D) Autoradiography of the NUB1L mutants shown in A after incubation with GST-FAT10 coupled GSH-sepharose, washing and separation by SDS-PAGE. Lane occupation is the same as in B. Only wild-type NUB1L, NUB1, and NUB1L∆UBL interact strongly, NUB1L∆UBA3 interacts weakly with GST-FAT10. The experiments were repeated three times with similar outcomes. 41
Chapter 1 covered proteins were separated by SDS-PAGE and analysed by autoradiography. As can be seen in figure 7A, C and 9C, only wild-type NUB1L, NUB1, and NUB1L∆UBL were able to pull down His6-FAT10 from the lysates. A subsequent immunoprecipitation of the supernatants with anti-His6 antibody demonstrated that sufficient His6-FAT10 was available in all reactions, so lack of expression can be excluded as a reason for the negative results. From the experiments presented in figure 6, figure 7 and figure 9C, D we conclude that the interaction of FAT10 and NUB1L depends on the presence of all three UBA-domains, but is indepen- dent of the UBL-domain of NUB1L. The deletion of only 14 amino acids in NUB1, which is about one third of the UBA2 domain is not sufficient to abrogate the interaction of NUB1 and FAT10. Figure 7: Co-immunoprecipitation of NUB1L mutants and FAT10. (A, C)Empty vector (mock) or HA-tagged NUB1L mutants lacking one or more UBA-domains were transfected either alone (- His-FAT10) or together with His 6-tagged FAT10 (+ His-FAT10) into HEK293T cells. The cells were lysed and an anti-HA immunoprecipitation was performed. The proteins bound by the antibody were separated by SDS-PAGE and analyzed by autoradiography. The NUB1L mutants appear between the 60 and the 90 kD molecular weight markers indicated at the right. An arrow on the right side of the gel denotes the position of co-immunoprecipitated FAT10. Only wild-type HA-NUB1L and HA-NUB1L∆UBL coprecipitated significant amounts of His 6-FAT10. (B, D) To analyze the amount of FAT10 available, the supernatants after the immunoprecipitation shown in A and C were precipitated with anti-His 6 antibodies, and the bound proteins separated by SDS-PAGE. Lane occupation is the same as in A and C, respectively. His 6- FAT10 was present in all FAT10 transfected cells. The experiments were repeated three times yielding similar results. 42
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: Chapter 1 no correlation between th
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 (
Page 91 and 92: 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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