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

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Introduction NUB1 has recently been implicated in two different types of neurodegenerative diseases, although the evidence is, admittedly, only circumstantial. NUB1 has been found to interact with AIPL1 (Aryl hydrocarbon receptor-interacting protein- like 1), a protein which is expressed solely in the rod photoreceptors of the adult human retina and is mutated in patients with Leber congenital amaurosis (LCA), a severe, early-onset form of retinal degeneration (Akey et al., 2002). AIPL1 func- tions as part of a chaperone heterocomplex containing both HSP70 and HSP90 (de Quintana et al., 2008) and is able to promote the translocation of NUB1 from the nucleus to the cytoplasm as well suppress the formation of NUB1 containing cytoplasmatic inclusions (van der Spuy and Cheetham, 2004). Interestingly, mutations in both the chaperone (de Quintana et al., 2008) as well as the NUB1 (Kanaya et al., 2004) binding sites have been found in patients with Leber congenital amaurosis. The second link to neurodegeneration derives from the interaction of NUB1 with synphilin-1, which is a major component of inclusion bodies found in the brains of patients with neurodegenerative α-synucleinopathies, such as Parkinson’s disease. It could be shown that NUB1 is able to suppress the formation of synphilin-1 containing inclusion bodies, presumably by proteasomal degradation of synphilin-1 (Tanji et al., 2006). The same study also demonstrated the presence of NUB1 in synphilin-1 positive Lewy bodies of Parkinson’s disease, while a follow-up study revealed NUB1 to only be present in inclusions found in patients with α-synucleinopathies, but not in inclusions observed in patients with other neurodegenerative diseases (Tanji et al., 2007). Through a yeast two-hybrid screen with the ubiquitin-like modifier FAT10 as a bait, NUB1L was recently identified as a non-covalent binding partner of FAT10, which could be verified both in vivo as well as in vitro. In addition, NUB1L was shown to specifically interact with FAT10 and failed to bind either ubiquitin or SUMO. Strikingly, this study was unable to reproduce the interaction with NEDD8, neither in vivo nor in vitro, although under the same conditions, FAT10 displayed robust interaction with NUB1L. Furthermore, even a 10-fold excess of NEDD8 was unable to compete with FAT10 for NUB1L binding. Overexpres- sion of NUB1L had no influence on the subcellular localization of FAT10. It did, however, lead to a marked increase in the rate of FAT10 degradation, which is consistent with its role as a putative proteasomal FAT10-receptor. (Hipp et al., 2004). 27
The Autophagy-Lysosome System Introduction Autophagy is an intracellular degradation system which delivers cytoplasmatic compontents to the lysosome. There are three distinct types of autophagy – microautophagy, chaperone-mediated autophagy and macroautophagy – and the name “autophagy” usually refers to the process of macroautophagy unless other- wise specified (Mizushima and Klionsky, 2007). Microautophagy involves the bud- ding of small cytosol-containing vessels directly into the lysosomal lumen. During chaperone-mediated autophagy, proteins carrying a signal-peptide for lysosomal sorting are directly transported into the lysosome by the transporter LAMP-2a, which is assisted by cytosolic and lysosomal chaperones of the HSC70 family (Massey et al., 2006). Macroautophagy is mediated by a unique organelle termed the autophagosome. During its process, a crescent-shaped isolation membrane forms de novo and encloses cytoplasmatic components – sometimes including whole organelles – in a double-membraned vesicle, which subsequently fuses with a lysosome for the degradation of its cargo and inner membrane (Ohsumi, 2001 and Fig. 5). Nutrient starvation – and the lack of amino acids in particular – is the most typi- cal trigger of autophagy, although a number of other factors inclucing, but not lim- ited to, insulin, growth factors or TRAIL are also able to negatively or positively affect autophagy (Mizushima, 2007). Many of the signals modulating autophagy converge at mTOR (mammalian target of rapamycin), which is a master regula- tor of nutrient signaling and potent inhibitor of autophagy. Inhibition of mTOR ultimately results in autophagosome formation, which is mediated through the concerted action of approximately 20 autophagy-related (ATG) proteins and involves the action of two ubiquitin-like conjugation systems. In a first step, the ubiquitin-like modifiers ATG8 – which requires prior processing by ATG4 to re- move its C-terminal extension – and ATG12 are activated through their common E1 enzyme ATG7. ATG8, which in mammals is known as LC3 (light chain 3), then becomes conjugated to the lipid phosphatidyl-ethanolamine in the forming isolation membrane through its E2 enzyme ATG3. Following completion of the autophagosome, ATG8 gets recycled from the outer autophagosomal membrane by deconjugation from the phospholipid, however, it remains attached to the inner membrane and this portion is degraded upon fusion with the lysosome. Owing to its prominent localization to the inside of the autophagosomal membrane, ATG8 has been suggested to function as an anchor for autophagy substrates. ATG12 in turn becomes conjugated to ATG5 through the action of its E2 enzyme ATG10. 28
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: Introduction Interestingly, a recen
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
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
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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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