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

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Chapter 1 Ortolan et al., 2000), and Rad23 inhibited the in vitro degradation of Ub5-DHFR (Raasi and Pickart, 2003). With respect to the function of UBA-UBL proteins in degradation it was proposed that they could operate as inhibitors of multiubiqui- tin chain assembly (Ortolan et al., 2000), inhibitors of deubiquitylation (Raasi and Pickart, 2003), linkers to the proteasome, and substrate carriers (Wilkinson et al., 2001). A careful investigation based on the reconstitution of Rad23- and Rpn10- deficient proteasomes with recombinant proteins and analysis of the degradation of different substrates clearly showed a substrate specificity of the UBL-UBA proteins beyond the modification with polyubiquitin (Verma et al., 2004a). This study demonstrated that deletion of a specific UBL-UBA protein lead to a slow down but not to a complete inhibition of the proteolysis of some substrates, but left others unaffected. However, in all cases the effect of the UBL-UBA protein was strictly dependent on the presence of the UBA-domain. Here we report the unexpected finding that the accelerated degradation of FAT10 by NUB1L is independent of all three of its UBA-domains. Since FAT10 and NUB1L∆UBA1-3 could not be co-immunoprecipitated, this enhancement in degradation does not depend on the interaction of both proteins. Moreover, NUB1L was able to accelerate the degradation of the two isolated UBL-domains of FAT10 when fused to GFP. Since only the N-terminal UBL-domain of FAT10 bound to NUB1L (Fig. 11), this is the second independent experiment in which we found no correlation between the ability of NUB1L to bind a FAT10 variant and to accelerate its degradation. Therefore, a role of NUB1L as a proteasomal receptor for FAT10 can not be the only mode of action. Because FAT10 degradation occurs also independently of ubiquitylation (Hipp et al., 2005), a role for NUB1L in the protection from deubiquitylation is unlikely as well. Also a protection from pu- tative FAT10-specific proteases is unlikely, since we failed to obtain evidence for their existence in two independent cell lines in the presence or absence of IFN-γ (Hipp et al., 2005). In our experiments, the ability of NUB1L to accelerate FAT10 degradation relied solely on the UBL domain-dependent interaction with the 26S proteasome, since both NUB1L and NUB1L∆UBA1-3 had a similar effect on the degradation rate of FAT10 while NUB1L∆UBL was ineffective (Fig. 8). Given that NUB1L and FAT10 can bind independently to the 26S proteasome (Fig. 13), we favour the hypothesis that binding of NUB1L to the 26S proteasome with its N-terminal UBL domain induces a conformational change in the 19S regulator that favours the binding and/or degradation of FAT10 and FAT10-conjugated proteins at an independent docking site. This scenario would be analogous to the one suggested by Verma et al. who showed that binding of the VWA domain of Rpn10 serves to facilitate the degradation of polyubiquitylated substrates delivered by 55
Chapter 1 Rad23 (Verma et al., 2004a). In order to address this hypothesis for NUB1L and FAT10, it will be important to map the binding site of FAT10 at the 26S proteasome. Curiously, the mutant FAT10-N-GFP is quite a stable protein (Fig. 12A), despite its ability to interact with the proteasome (Fig. 13D). Degradation of FAT10-N- GFP, however, can be readily induced by coexpression of NUB1L. These results combined argue against the recently proposed model that mere binding to the proteasome is sufficient to target for proteasomal degradation (Janse et al., 2004). Verma et al. found that the polyubiquitylated proteasome substrate Sic1 bound to Rpn10-deficient 26S proteasomes in dependency of Rad23, but no degradation was observed (Verma et al., 2004a). Thus binding seems to be a necessary but not a sufficient prerequisite for proteasomal degradation of at least some substrates. For the proteolysis of such substrates like Sic1 or FAT10, the binding of their respective facilitators Rpn10 or NUB1L to the proteasome appears to be important. Experimental Procedures Antibodies. The anti-HA mAb clone HA7 (Sigma) was used for immunoprecipitation in figures 7, 8, 9, 10, 11, 12 and for the Western blot in figure 13A. The anti-His6 mAb clone BMG His-1 (Roche) was used for immunoprecipitation shown in figures 7B, D and 9D. A mixture of monoclonal anti-GFP clones 7.1 and 13.1 (Roche) was used for the immunoprecipitation in figure 11A and for the western blot in figure 13B. An anti-HA mAb clone HA7 antibody peroxidase-conjugate (Sigma) was used for the western blot shown in figure 13C, and a polyclonal rabbit anti-GFP antibody (Sigma) for the western blot in figure 13D. The western blots presented in figure 13E and F were performed with a rabbit polyclonal anti-Rpt6 (S8) from Biomol. The immunoprecipitation of the proteasome was performed with 5 µl of ascitis fluid of the anti-HN3 antibody MCP444 (Hendil et al., 1995). The mouse monoclonal anti-iota antibody 27K was kindly provided by Dr. Klaus Scherrer (Paris). Anti-FAT10 antibody has been described (Hipp et al., 2005), anti-NUB1 antibody was a gift from Dr. Michael E. Cheetham (London). The anti-GAPDH antibody was purchased from Ambion. All antibodies were cou- pled to EZview Red Protein A or G Affinity Gel (Sigma) for immunoprecipitation. All horseradish peroxidase-conjugated secondary antibodies were purchased from DAKO. 56
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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 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: Chapter 1 domains of NUB1L is requi
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
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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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