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

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Introduction Figure 2: The ubiquitylation pathway. Free ubiquitin (Ub) is activated in an ATPdependent manner with the formation of a thiol-ester-linkage between E1 and the carboxyl terminus of ubiquitin. Ubiquitin is then transferred to one of a number of different E2s. E2s associate with E3s, which may have substrate already bound. For HECT domain E3s, ubiquitin is next transferred to the active-site cysteine of the HECT domain followed by transfer to substrate(s) (as shown) or to a substrate-bound polyubiquitinchain. For RING E3s, current evidence indicates that ubiquitin is transferred directly from the E2 to the substrate. (Modified from Fang and Weissman, 2004). turned out to indeed be the most prevalent function of ubiquitin conjugation. Pro- teasomal targeting of proteins is mediated by the attachment of a chain of four or more K48-linked ubiquitin moieties (Thrower et al., 2000). In a limited number of cases, the attachment of a K29-linked chain also leads to proteasomal degradation (Johnson et al., 1995), however, most other variations of ubiquitin attachment are means to a different end. K63-linked ubiquitin displays a distinct chain topology and is involved in signal transduction (Krappmann and Scheidereit, 2005) and DNA repair (Friedberg et al., 2005). In some instances, modification of the target protein with a single ubiquitin moiety (monoubiquitylation), or with several separate ubiquitins at different lysines (multiubiquitylation) is sufficient to modify a protein’s activity or to create a new binding site. As such, monoubiquitylation has been implicated in endocytosis, vesicular sorting, histone regulation and the budding of retroviruses (Hicke, 2001; Di Fiore et al., 2003). Substrate specificity of the ubiquitin system is conferred by a multitude of E3 enzymes, which are responsible for recognizing their specific target protein. In genomics studies, several hundered putative E3 candidate genes have been identified. E3 ubiquitin-protein ligases are defined as enzymes which bind, through direct interaction or with the help of additional adaptor proteins, specific protein substrates and facilitate the transfer of ubiquitin from a thiolester intermediate of their cognate E2 enzyme to an isopeptide linkage with the target protein. The 15
Introduction majority of ubiquitin-protein ligases fall into one of two subcategories, the HECT- domain E3s or the RING-finger E3s (Fang and Weissman, 2004). The HECT (Homologous to E6-AP C-terminus)-domain family was the first family of ubiquitin-ligases to be identified. The first member of this family, E6-AP, was discovered as a cellular protein which was required for the ubiquitylation and sub- sequent degradation of the p53 tumor suppressor by the human pappilomavirus E6 oncoprotein (Scheffner et al., 1994). HECT-domain E3s first transfer ubiquitin from the bound E2 onto their active site cysteine in the C-terminal HECT-domain before they pass it on to their substrate protein. The RING (Really Interesting New Gene)-domain family is the largest family of ubiquitin-ligases. The RING-finger is defined by eight conserved cysteines and histidines which together coordinate two zinc ions (Borden and Freemont, 1996). The first member of this family which was identified is the ubiquitin-ligase E3α, which is responsible for ubiquitylating substrates of the N-end rule pathway. RING-domain E3s do not transfer the activated ubiquitin onto themselves, but rather facilitate its transfer from the tightly bound E2 directly onto the substrate protein. This family of E3s consists of both single subunit enzymes, which contain all the necessary domains in one protein, for example Cbl, as well as large multi- meric complexes such as the SCF-complex, or the anaphase-promoting-complex, in which the RING domain exists as a separate subunit (Joazeiro and Weissman, 2000). The other two subfamilies are relatively small and both contain variants of the RING-domain. The PHD-finger was first identified as a component of viral ubiquitin-ligases, but has subsequently also been found in mammalian proteins (Coscoy and Ganem, 2003). The U-box, in turn, is a modified RING-domain with no coordinated zinc ions. The ubiquitin-ligase CHIP for example, which functions as an E3 for HSP90 interacting proteins, is a member of this last family (Jiang et al., 2001). In contrast to the large number or E3 enzymes, the mammalian genome encodes for only about 30 different E2 ubiquitin-conjugating enzymes (von Arnim, 2001). As a consequence, each E2 enzyme is responsible for serving a number of different E3s. All known E2 enzymes belong to the same family, which is characterized by the presence of a catalytic domain called the UBC-domain. Some E2s possess additional C-terminal or N-terminal extensions, which are responsible for medi- ating subcellular localization or recognition of E3 enzymes. With a few exceptions 16
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: Introduction are the major source o
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
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Chapter 2 Figure 14: Purified compo
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Chapter 2 evaluation of the NUB1L b
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Chapter 2 teins which contain intri
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Chapter 2 Combined with our studies
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Chapter 2 Recombinant expression of
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Chapter 2 et al., 2003), both at a
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Abstract Chapter 3 During misfolded
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Chapter 3 tion between the UPS and
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Chapter 3 Figure 18: HDAC6 interact
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Chapter 3 Figure 19: Both the BUZ d
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Chapter 3 of HDAC6 was required for
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Chapter 3 precipitates with HDAC6 (
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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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