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NEDD8-CHOP-Reporter (DeNEDDylation Assay Kit)

Catalog Number
PR1004

Product Information

Price

$995.00 USD

Benefits
Components
Suggested Uses
References

Documentation

Manual
Limited Use Label License

Product Description

 

Ubiquitin and Ubiquitin Like Proteins

In cells, proteins are tagged for degradation by ubiquitin and sent to the proteasome. In contrast, covalent modification of cellular proteins by the ubiquitin-like modifier SUMO (small ubiquitin-like modifier) regulates various cellular processes, such as nuclear transport and signal transduction. The ubiquitin family of proteins fall into two classes: the first class, ubiquitin-like proteins (UBLs) function as modifiers in a manner analogous to that of ubiquitin. Examples of UBLs are SUMO, Nedd8 (also called Rub1), ISG15, Apg8, Apg12, and Fat10. Proteins of the second class include parkin, RAD23 and DSK2, are designated ubiquitin-domain proteins (UDPs). These proteins contain domains that are related to ubiquitin but are otherwise unrelated.
 

Conjugation Machinery

The conjugation of Ub/UBLs to target proteins requires an orchestrated addition of Ub/UBLs to lysine residues in the target protein by E1 (activating enzyme), E2 (conjugating enzyme), and E3 (ligase) in an ATP dependent manner. The enzymes form an isopeptide bond between the carboxy-terminus of the UBL and the e-amino group of the lysine residue of target proteins. 
 

DeNEDDylating Enzymes

NEDDylation is a reversible process in which deconjugation is performed in cells by deNEDDylating enzymes, otherwise known as isopeptidases. Isopeptidases are cysteine proteases that can be divided into multiple familes. The roles of isopeptidases include recycling of fused ubiquitin/UBL and processing pro-ubiquitin/UBL by cleavage to the mature form. Removal of ubiquitin or UBL moieties can affect cellular physiology in a number of ways, and several isopeptidases have been linked to pathologies such as cancer and cardiovascular disease.

About the Assay

The NEDD8-CHOP-Reporter DeNEDDylation Assay consists of NEDD8 fused to a reporter enzyme and the reporter enzyme substrate. Upon conjugation of NEDD8, the reporter is rendered catalytically inactive. Following cleavage of the NEDD8-reporter system by isopeptidase activity, the activated, free reporter subsequently acts upon its substrate. Thus, in the coupled assay, the signal generated by cleavage of the reporter enzyme’s substrate is a quantitative measure of isopeptidase activity.

 

Benefits

  • Superior to Nedd*-AMC or FRET-based assays

  • Rapid and robust readout for deneddylating activity within 60 minutes; signal to noise ratio >20

  • Non-radioactive reporter substrates

  • Miniaturization to multiwell format; cost effective screening

  • Assay tests deconjugating activity between ubiquitin/UBL and a physiologically relevant protein

  • Reporter system does not contain chemically reactive groups (like Nedd*-AMC) that could give false positives in screening

Components

  1. NEDD8-CHOP-Reporter

  2. DEN1 (Deneddylation Enzyme, Control)

  3. Reporter Substrate

Suggested Uses

  •  Demonstrate novel deneddylating (de-NEDD8) activity

  • High throughput screening for inhibitors or activators of deneddylating (de-NEDD8) activity

References

Arnold, J.J., Bernal, A., et al. (2005). “Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity.” Anal Biochem, Nov 17.

Chung, C. H. and S. H. Baek (1999). "Deubiquitinating enzymes: their diversity and emerging roles." Biochem Biophys Res Commun 266(3): 633-40.

Ciechanover, A. (2001). "Ubiquitin-mediated degradation of cellular proteins: why destruction is essential for construction, and how it got from the test tube to the patient's bed." Isr Med Assoc J 3(5): 319-27.

Ciechanover, A. (2003) "The ubiquitin proteolytic system and pathogenesis of human diseases: a novel platform for mechanism-based drug targeting." Biochem Soc Trans 31(2): 474-81.

D'Andrea, A. and D. Pellman (1998). "Deubiquitinating enzymes: a new class of biological regulators." Crit Rev Biochem Mol Biol 33(5): 337-52.

Dang, L. C., F. D. Melandri, et al. (1998). "Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes." Biochemistry 37(7): 1868-79.

Hall, J., Mattern, M., and Butt, T. (2005). “Mining the ubiquitin pathway.” The Scientist 19 (23): 42-43.

Hershko, A. and A. Ciechanover (1998). "The ubiquitin system." Annu Rev Biochem 67: 425-79.

Hochstrasser, M. (1996). "Ubiquitin-dependent protein degradation." Annu Rev Genet 30: 405-39.

Hochstrasser, M. (2002). "Molecular biology. New proteases in a ubiquitin stew." Science 298(5593): 549-52.

Nicholson, B, Leach, C.A., et al (2008). “Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities.” Prot. Sci., in press

Wilkinson, K. D. (1997). "Regulation of ubiquitin-dependent processes by deubiquitinating enzymes." Faseb J 11(14): 1245-56.

Wilkinson, K. D. (2000). "Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome." Semin Cell Dev Biol 11(3): 141-8.