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

More documents

Recommendations

Info

Chapter 1 Figure 10: Treatment with IFN-γ and TNF-α upregulates NUB1L expression and accelerates the degradation of FAT10. (A) Anti-NUB1L Western blot of lysates from mock transfected and HA-NUB1 transfected HEK293T cells (top panel), Western analysis of GAPDH protein is used as loading control (bottom panel). (B) Anti-NUB1L Western blot of lysates from untreated HeLa cells and HeLa cells induced with IFN-γ and TNF-α for one day. An arrow at the right indicates the position of NUB1L (top panel); the same lysates probed with anti-GAPDH antibodies serves as loading control (bottom panel). (C) Real time RT-PCR analysis of FAT10 and NUB1L mRNA expression in HeLa cells before (-) and after (+) treatment with TNF-α and IFN-γ for one day. (D) HeLa cells were transfected with an HA-FAT10 expression construct and the degradation rate of FAT10 was determined in a pulse-chase experiment. K- denotes an IP with irrelevant antibody. (E) Analysis of the degradation rate of endogenous FAT10 after induction of FAT10 and NUB1L by IFN-γ and TNF-α. The pulse-chase experiment was performed as in E using an anti-FAT10 antibody. K- is an IP with pre-immune serum. The position of FAT10 is indicated by an arrowhead. (F) Graphic evaluation of the different degradation profiles of FAT10 as determined in D (squares) and E (triangles). 47
Chapter 1 ing SUMO-1-GFP or the C-terminal UBL-domain of FAT10 (FAT10-C-GFP). Fig- ure 11B demonstrates that the amounts of NUB1L available were sufficient in all co-expression reactions to allow detection if bound to the respective GFP fusion protein. Figure 11: The N-terminal-, but not the C-terminal UBL-domain of FAT10 interacts with NUB1L. (A) Empty vector (mock), the C-terminal UBL-domain of FAT10 (FAT10-C), the N-terminal UBL-domain of FAT10 (FAT10-N), wild-type FAT10 as positive- and SUMO-1 as negative control, all as GFP-fusions, were transfected either alone (- HA-NUB1L) or together with HA-tagged NUB1L (+ HA-NUB1L) into HEK293T cells. The cells were lysed and an anti-GFP immunoprecipitation was performed. The proteins bound by the antibody were separated by SDS-PAGE and analyzed by autoradiography. An arrow denotes the position of NUB1L. Molecular weight markers are shown at the right. (B) To analyze the amount of NUB1L available, the supernatants after the immunoprecipitation shown in A were precipitated with anti-HA antibodies, and the bound proteins separated by SDS-PAGE. Lane occupation is the same as in A. An arrow denotes the position of NUB1L. HA-NUB1L was present in all HA-NUB1L transfected cells. The experiments were repeated two times with similar outcome. NUB1L accelerates both FAT10-N-GFP and FAT10-C-GFP degradation To analyse the impact of NUB1L expression on the half-lives of the two isolated UBL-domains of FAT10, we first determined the rate of degradation of FAT10- N-GFP and FAT10-C-GFP alone. As can be seen in figure 12A and B, the degradation rates of the two UBL-domain-GFP fusion proteins in HEK293T cells were significantly different from each other. FAT10-C-GFP was degraded with a rate comparable to that of wild-type FAT10-GFP (Hipp et al., 2005) but slower than FAT10 itself (compare Fig. 8E and Fig. 12I). FAT10-N-GFP, in comparison, is a 48
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: Chapter 1 Treatment with IFN-γ and
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
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
Page 119 and 120:
Chapter 5 Figure 36: The antibody s
Page 121 and 122:
Discussion 30 years ago, ubiquitin
Page 123 and 124:
Discussion monomeric ubiquitin as w
Page 125 and 126:
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
Page 143 and 144:
Abbreviations aa amino acid AAA ATP
Page 145 and 146:
Amino Acids Alanine Ala A Arginine
show all

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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?