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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Strategies for Monitoring "Ubiquitin C-Terminal Hydrolase 1" (Yuh1) Activity.
Methods Mol Biol
December 2022
The Faculty of Natural Sciences, University of Haifa, Haifa, Israel.
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.
View Article and Find Full Text PDFMutant ubiquitin (UBB+1) associated with neurodegenerative disorders is hydrolyzed by ubiquitin C-terminal hydrolase L3 (UCH-L3).
FEBS Lett
August 2011
Department of Neuroscience, Faculty of Health Medicine and Life Sciences, Maastricht University, Maastricht, The Netherlands.
Mutant ubiquitin (UBB(+1)) accumulates in the hallmarks of tauopathies and polyglutamine diseases. We show that the deubiquitinating enzyme YUH1 of Saccharomyces cerevisiae and its mouse and human ortholog UCH-L3 are able to hydrolyze the C-terminal extension of UBB(+1). This yields another dysfunctional ubiquitin molecule (UB(G76Y)) with biochemical properties similar to full length UBB(+1).
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Biochem Biophys Res Commun
December 2009
Kyoto University, Research Reactor Institute, Kumatori, Osaka Japan.
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View Article and Find Full Text PDFUbiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation.
Biochemistry
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Laboratory of Cellular and Molecular Biology, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)C(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1.
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