Strategies for Monitoring "Ubiquitin C-Terminal Hydrolase 1" (Yuh1) Activity
Shahaf Saad1,2, Eden Berda1,2, Yuval Klein1
1The Faculty of Natural Sciences, University of Haifa, Haifa, Israel.
Methods in Molecular Biology (Clifton, N.J.)
|November 29, 2022
Summary
We developed a new method to test the activity of ubiquitin C-terminal hydrolase Yuh1 (Yuh1) in yeast and bacteria. This assay monitors the trimming of modified ubiquitin and NEDD8 proteins, crucial for cellular regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ubiquitin C-terminal hydrolases (UCHs) are enzymes that cleave ε-linked amide bonds at the C-terminus of ubiquitin.
- UCHL3 is a conserved UCH enzyme with dual functionality, recognizing both ubiquitin and NEDD8 modifiers.
- Yuh1 is the sole UCH family member in Saccharomyces cerevisiae and recognizes both ubiquitin and its paralogue, Rub1.
Purpose of the Study:
- To establish and validate a novel method for assessing the enzymatic activity of Yuh1.
- To enable the study of Yuh1 function in both bacterial and yeast expression systems.
Main Methods:
- Bacterial and yeast expression of Yuh1.
- Monitoring C-terminal trimming of modified ubiquitin (UBB+1) and Rub1 (Rub1+1) substrates.
- Utilizing immunoblotting and AMC fluorescence assays for activity detection via plate reader.
Main Results:
- The described method successfully monitors Yuh1 activity by detecting the C-terminal trimming of UBB+1 and Rub1+1.
- Increased AMC fluorescence correlates with Yuh1 enzymatic activity, providing a quantitative readout.
- The method is applicable to Yuh1 expressed in both bacterial and yeast systems.
Conclusions:
- A robust and versatile method for assaying Yuh1 enzymatic activity has been developed.
- This method facilitates the study of ubiquitin and NEDD8 C-terminal processing in cellular contexts.
- The findings contribute to understanding UCH enzyme function and regulation.
