A Method for Assaying Deubiquitinating Enzymes-1
| Abstract |
A general method for the assay of deubiquitinating enzymes was described in detail using 125I-labeled ubiquitin-fused αNH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. Since the tyrosine residue in the PESTc portion of the fusion protein was almost exclusively radioiodinated under a mild labeling condition, such as using IODO-BEADS, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid soluble products. Using this assay protocol, we could purify six deubiquitinating enzymes from chick skeletal muscle and yeast and compare their specific activities. Since the extracts of E. coli showed little or no activity against the substrate, the assay protocol should be useful for identification and purification of eukaryotic deubiquitinating enzymes cloned and expressed in the cells.
| Introduction |
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| Abstract Introduction Materials and Methods Results and Discussion References |
Ubiquitin (Ub) is a highly conserved 76 amino acid protein found in all eukaryotic cells (1,2). Ub is covalently ligated to a variety of intracellular proteins through an isopeptide linkage. Ubs by themselves or that have already been conjugated to proteins may also be ligated to additional Ub molecules to form branched poly-Ub chains. This ubiquitination has been implicated in the regulation of diverse cellular processes, such as selective protein breakdown, cell cycle regulation, and stress response (3-5). In addition, protein ubiquitination in vivo is a dynamic, reversible process that is under control responding to external stimuli, such as heat shock and starvation (6-8). Therefore, the enzymes that proteolytically remove Ub from Ub-protein conjugates should be of importance in maintaining the steady-state levels of free Ub for its diverse cellular functions.
Ubs are encoded by two distinct gene classes. One is a poly-Ub gene that encodes a poly-protein of tandemly repeated Ubs (9,10). The other encodes a fusion protein in which a single Ub is linked to a ribosomal protein consisting of 52 or 76-80 amino acids. The transient association of Ub with the ribosomal proteins has been suggested to promote their incorporation into ribosomes (11). Therefore, proteolysis at the peptide bonds between Ub and the extension proteins is required for generation of ribosomal proteins for ribosome biogenesis as well as of free Ubs.
Deubiquitinating enzymes (DUBs) are known to consist of a large protein family in eukaryotes. For example, the budding yeast has 17 genes for DUBs (4). Moreover, so far more than 60 full-length DUB sequences have been identified in eukaryotes (12). However, comprehensive searches for DUBs, particularly in mammalian cells, were hampered due to the lack of rapid and efficient methods for assaying the enzymes. We have recently reported that 125I-labeled Ub-PESTc serves as an excellent substrate for the sensitive and quantitative assay of various DUBs (13,14). Using this assay, we have also isolated a number of novel DUBs in chick skeletal muscle and yeast (13, 15-18). Here we describe the detailed protocol for assaying DUBs using 125I-labeled Ub-PESTc. We also compare the sensitivity of this method to that using a fluorogenic peptide substrate, carbobenzoxy-LRGG-7-amido-4-methylcoumarin (Cbz-LRGG-AMC), that has also been used as a substrate for DUBs (19).
| Materials and Methods |
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| Abstract Introduction Materials and Methods Results and Discussion References |
Materials
Yeast Ub hydrolase-1 (YUH1) was purified as described previously (20). The purified Ub-specific protease-6 (yUBP-6) in yeast and cUBP41, Ub C-terminal hydrolase-1 (cUCH-1), cUCH-6, and cUCH-8 in chick skeletal muscle were prepared as described (13, 15-18). Ub-PESTc was purified from an E. coli strain AR13 carrying pNMHUB-PESTc as described by Yoo et al. (21). Ub-aldehyde was prepared as described (13). Cbz-LRGG-AMC was kindly provided by Dr. K. Tanaka (Tokyo Metropolitan Institute of Medical Science, Japan).
Radioiodination of Ub-PESTc
Ub-PESTc was radiolabeled with Na125I using IODO-BEADS (Pierce) by following the procedure recommended by the manufacturer and Markwell (22). One IODO-BEAD was incubated in 0.2 ml of 0.1 M Tris-HCl (pH 7) in a microfuge tube for 5 min at room temperature. After incubation, 0.2 mg of the purified Ub-PESTc and 200 μCi of Na125I were added to the tube and further incubated for the next 15 min. The radioiodinated Ub-PESTc (i.e., the liquid portion) was then removed and subjected to gel filtration on a Sephadex G-10 column equilibrated with the Tris buffer to remove free iodine. Upon the iodination procedure, approximately 85% of 125I was incorporated into Ub-PESTc.
Assay for hydrolysis of 125I-labeled Ub-PESTc
Reaction mixtures (final 0.1 ml) contained proper amounts of the purified DUBs, 10-20 μg of 125I-labeled Ub-PESTc, 0.1 M Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol, and 5% (v/v) glycerol. After incubation of the mixtures for appropriate periods at 37 °C, the reaction was terminated by adding 50 μl of 40% (v/v) trichloroacetic acid and 50 μl of 1.2% (w/v) bovine serum albumin. The samples were vortexed and centrifuged for 10 min at 10,000 x g using a microfuge, and aliquots (0.1 ml) of the resulting supernatants were counted for radioactivity using a gamma-counter (13).
Assay for hydrolysis of Cbz-LRGG-AMC
Reaction mixtures (final 0.1 ml) contained appropriate amounts of the purified DUBs, 0.2 mM Cbz-LRGG-AMC, 0.1 M Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol, and 5% (v/v) glycerol. After incubation of the mixtures for various periods at 37 °C, the reaction was terminated by adding 0.1 ml of 1% (w/v) SDS and 0.8 ml of H2O. Release of free AMC by the enzyme reaction was determined by measuring its fluorescence at 380 nm (excitation) and 440 nm (emission) (23). Proteins were quantified as described by Bradford (24).
Gel electrophoresis
Polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol was performed using Tris-Tricine buffer as described by Schägger and von Jagow (25). The discontinuous slab gels contained 4, 10 and 16% polyacrylamide to improve resolution of small proteins. The sample buffer contained 150 mM Tris-HCl (pH 6.8), 1.5% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 0.002% (w/v) bromophenol blue and 7% (v/v) glycerol. After electrophoresis, the gels were stained with Coomassie blue R-250 or subjected to autoradiography.
| Results and Discussion |
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| Abstract Introduction Materials and Methods Results and Discussion References |
Rechsteiner and coworkers (21) have constructed Ub-αNH-peptide extensions containing "PEST" sequences. Of these, Ub-PESTc contains a peptide extension of 18 amino acids, which carries a single tyrosine residue that can be radioiodinated and is short enough to be released as an acid-soluble product when incubated with DUBs. Moreover, they have reported that the recombinant Ub-PESTc protein can be easily purified by heating, such as at 85 °C, because fusion of the short peptide to Ub does not alter the heat resistance of Ub molecule. In addition, they have demonstrated that Ub-PESTc appears correctly processed to yield free Ub upon incubation with the chromatographic fractions of rabbit reticulocytes. Therefore, we chose Ub-PESTc for labeling with 125I and hence for using the labeled protein as a substrate for the assay of DUBs. Overall procedure for the enzyme assay is summarized in Fig. 1
Fig. 1: [Enlarge] | Schematic representation for a method for assaying DUBs using 125I-Ub-PESTc. |
By following the protocol, we determined the activity of YUH1 by measuring its ability to release radioactive PESTc into an acid-soluble form from 125I-labeled Ub-PESTc. The purified enzyme was incubated with the radioiodinated substrate for various periods in the absence and presence of Ub-aldehyde, which is known as a specific inhibitor of DUBs (26). Table 1 shows that the acid-soluble radioactivity increases in an incubation time-dependent fashion and this increase can be completely blocked by the treatment of Ub-aldehyde. Since YUH1 as well as other DUBs are known to specifically cleave the carboxyl side of the C-terminal Gly residue of Ub, the acid-soluble products should represent the PESTc peptide released from the Ub-peptide extension.
