Abstract
The family of ubiquitin C-terminal hydrolases (UCHs(releases ε-linked amide bonds positioned at the C-terminus of ubiquitin. UCHL3 is a highly conserved and dual functional member of this family, recognizing C-terminal extensions of two paralogous modifiers: ubiquitin and NEDD8. The Saccharomyces cerevisiae orthologue of UCHL3, namely, Yuh1, is the only UCH family member in this organism. Like UCHL3, Yuh1 recognizes ubiquitin as well as Rub1, the direct orthologue of NEDD8 in S. cerevisiae. We describe here a method for examining the activity of bacteria and yeast expressed Yuh1 by monitoring the C-terminal trimming of UBB + 1 and Rub1 + 1 through immunoblotting and the increased AMC fluorescence readout detected through a plate reader.
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Bibliography
Geurink PP, van der Heden van Noort GJ, Mulder MPC et al (2019) Profiling DUBs and Ubl-specific proteases with activity-based probes. Methods Enzymol 618:357–387. https://doi.org/10.1016/bs.mie.2018.12.037
Hermanns T, Pichlo C, Woiwode I et al (2018) A family of unconventional deubiquitinases with modular chain specificity determinants. Nat Commun 9(1):799. https://doi.org/10.1038/s41467-018-03148-5
Hermanns T, Pichlo C, Baumann U et al (2022) A structural basis for the diverse linkage specificities within the ZUFSP deubiquitinase family. Nat Commun 13(1):401. https://doi.org/10.1038/s41467-022-28049-6
Misaghi S, Galardy PJ, Meester WJ et al (2005) Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate. J Biol Chem 280(2):1512–1520. https://doi.org/10.1074/jbc.M410770200
Johnston SC, Riddle SM, Cohen RE et al (1999) Structural basis for the specificity of ubiquitin C-terminal hydrolases. EMBO J 18(14):3877–3887
Linghu B, Callis J, Goebl MG (2002) Rub1p processing by Yuh1p is required for wild-type levels of Rub1p conjugation to Cdc53p. Eukaryot Cell 1(3):491–494
Mendoza HM, Shen L-n, Botting C et al (2003) NEDP1, a highly conserved cysteine protease that deNEDDylates Cullins. J Biol Chem 278(28):25637–25643
Pick E (2020) The necessity of NEDD8/Rub1 for vitality and its association with mitochondria-derived oxidative stress. Redox Biol 37:101765. https://doi.org/10.1016/j.redox.2020.101765
Sinha A, Israeli R, Cirigliano A et al (2020) The COP9 signalosome mediates the Spt23 regulated fatty acid desaturation and ergosterol biosynthesis. FASEB J 34:4870–4889. https://doi.org/10.1096/fj.201902487R
Yu Z, Kleifeld O, Lande-Atir A et al (2011) Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome. Mol Biol Cell 22(7):911–920. https://doi.org/10.1091/mbc.E10-08-0655
Scott DC, Monda JK, Grace CR et al (2010) A dual E3 mechanism for Rub1 ligation to Cdc53. Mol Cell 39(5):784–796. https://doi.org/10.1016/j.molcel.2010.08.030
Dennissen FJ, Kholod N, Hermes DJ et al (2011) Mutant ubiquitin (UBB+1) associated with neurodegenerative disorders is hydrolyzed by ubiquitin C-terminal hydrolase L3 (UCH-L3). FEBS Lett 585(16):2568–2574. https://doi.org/10.1016/j.febslet.2011.06.037
Chadwick L, Gentle L, Strachan J et al (2012) Review: unchained maladie - a reassessment of the role of Ubb(+1) -capped polyubiquitin chains in Alzheimer's disease. Neuropathol Appl Neurobiol 38(2):118–131. https://doi.org/10.1111/j.1365-2990.2011.01236.x
Lindsten K, de Vrij FM, Verhoef LG et al (2002) Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation. J Cell Biol 157(3):417–427. https://doi.org/10.1083/jcb.200111034
Krutauz D, Reis N, Nakasone MA et al (2014) Extended ubiquitin species are protein-based DUB inhibitors. Nat Chem Biol 10(8):664–670. https://doi.org/10.1038/nchembio.1574
Hermanns T, Hofmann K (2019) Bacterial DUBs: deubiquitination beyond the seven classes. Biochem Soc Trans 47(6):1857–1866. https://doi.org/10.1042/BST20190526
Acknowledgments
We thank Michael Glickman for providing us with plasmids. Studies at the Pick lab are supported by the Israel Science Foundation (ISF) grant no. 192/20 and Bet Margolin fellowships for E. B. and S. S. The authors would like to acknowledge the networking support by the ProteoCure COST action (CA20113).
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Saad, S., Berda, E., Klein, Y., Issa, S., Pick, E. (2023). Strategies for Monitoring “Ubiquitin C-Terminal Hydrolase 1” (Yuh1) Activity. In: Rodriguez, M.S., Barrio, R. (eds) The Ubiquitin Code. Methods in Molecular Biology, vol 2602. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2859-1_8
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DOI: https://doi.org/10.1007/978-1-0716-2859-1_8
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