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

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Chapter 5 FAT10 (Chiu et al., 2007) have all been described previously. GFP-LC3B has been contributed by Trond Lamark (unpublished data). GST-mFAT10 was gener- ated by amplifying the murine fat10 cDNA by PCR and cloning it into the vector pGEX2-TK through its EcoRI and XbaI restriction sites (Neha Rani, unpublished results). Generation of monoclonal antibodies. Monoclonal antibodies were gener- ated in essence as previously described (Yokoyama et al., 2006). Briefly, two mice were each immunized with 100 µg of recombinant GST-FAT10 in lipopeptide. Af- ter three rounds of booster immunizations, the animals were sacrificed and their spleens were harvested and a single cell suspension was prepared. Spleen cells were then fused with SP2/0-Ag14 myeloma cells at a 1:1 ratio and plated into flat- bottom 96-well plates. After selection with HAT medium, the surviving cells were screened by ELISA for the production of GST-FAT10 specific antibodies. A GST ELISA was performed to exclude those hybridomas which produced antibodies with anti-GST reactivity. The resulting hybridomas were then subjected to three rounds of subcloning. After expansion, antibodies were purified from hybridoma supernatants using a protein G column (GE Healthcare). ELISA. Antisera and hybridoma supernatants were screened by ELISA essen- tially as previously described (Hornbeck, 1992). Briefly, Microlon ELISA-plates (Greiner Bio-One) were coated with 20 µg/ml of recombinant protein. Antisera were diluted 1:25 or 1:50 in blocking buffer, hybridoma supernatants were diluted 1:2 in blocking buffer. Bound antibodies were detected with alkaline phosphatase- coupled protein G (Calbiochem) diluted 1:2500 in blocking buffer and developed with 3 mM p-nitrophenyl phosphate (Fluka). Substrate hydrolysis was measured at 405 nm wavelength in a Tecan microplate reader. Antibody isotypes were deter- mined using the Mouse Immunoglobulin Isotyping ELISA kit from BD Biosciences according to the manufacturer’s instructions. 119
Discussion 30 years ago, ubiquitin was first identified as a small protein capable of targeting cellular proteins for degradation through its covalent attachment (Ciechanover et al., 1978, 1980; Hershko et al., 1980). Later studies revealed it to be involved in the degradation of the majority of intracellular proteins in eukaryotes – and identified the 26S proteasome as the protease responsible for the proteolytic destruction of its target proteins (Hershko and Ciechanover, 1998). Polyubiquity- lated proteins can either bind directly to the proteasome (Deveraux et al., 1994; Lam et al., 2002), or they can be indirectly delivered via linker proteins such as hHR23/Rad23 or hPLIC/Dsk2 (Chen and Madura, 2002; Funakoshi et al., 2002). In addition, evidence has accumulated during the past few years that, under conditions where the proteasome ceases to function, alternative adaptor proteins such as HDAC6 or p62 can instead target these substrates for lysosomal degradation via the autophagic pathway (Kawaguchi et al., 2003; Iwata et al., 2005; Shin, 1998; Bjørkøy et al., 2005). Although ubiquitin was long thought to be the only protein capable of directly targeting others for destruction through its covalent attachment, the ubiquitin- like modifier FAT10 has recently emerged as cytokine-inducible alternative to ubiquitin-mediated proteasomal degradation. Interestingly, despite the fact that FAT10 can mediate the degradation of its substrates independently of ubiqui- tylation (Hipp et al., 2005 and chapter 2), its modus operandi is in many ways analogous to that of ubiquitin. Like ubiquitin, FAT10 can become covalently conjugated to its target proteins through its C-terminal glycine residue (Raasi et al., 2001) and can directly interact with the 26S proteasome. Alternatively, FAT10 can also be tethered to the proteasome through interaction with the UBL- UBA domain protein NUB1L, which – in addition to its role as an alternative proteasomal receptor for FAT10 – also functions as a facilitator of FAT10 degradation (chapter 1). As could be demonstrated for some polyubiquitylated substrates, mere physical attachment of a model FAT10-conjugate to the proteasome was insufficient to promote its degradation, which was instead dependent on the 120
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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
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Introduction majority of ubiquitin-
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Introduction example contained in t
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Introduction two modifiers apart fr
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Introduction Figure 4: Comparison o
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Introduction dependent apoptosis in
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Introduction Interestingly, a recen
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The Autophagy-Lysosome System Intro
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Introduction Figure 5: Molecular ma
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Introduction The transport of misfo
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Introduction where the proteasome i
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Chapter 1 The UBA domains of NUB1L
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Introduction Chapter 1 Ubiquitin, a
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Chapter 1 no correlation between th
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Chapter 1 covered proteins were sep
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Chapter 1 Figure 8: Accelerated deg
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Chapter 1 Treatment with IFN-γ and
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Chapter 1 ing SUMO-1-GFP or the C-t
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Chapter 1 Figure 12: Accelerated de
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Chapter 1 Figure 13: Co-immunopreci
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Chapter 1 domains of NUB1L is requi
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Chapter 1 Rad23 (Verma et al., 2004
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Chapter 1 FAT10-GFP expressing vect
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Chapter 2 Degradation of FAT10 by t
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Introduction Chapter 2 The proteaso
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Chapter 2 From our previous data it
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Chapter 2 Figure 14: Purified compo
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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
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Page 87 and 88: Chapter 3 of HDAC6 was required for
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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: Chapter 5 Figure 36: The antibody s
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
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