Activated USP14 DUB Assay Kit

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The Activated USP14 DUB Assay Kit contains all the reagents required to perform a USP14 deubiquitylase (DUB) assay. It comprises: USP14 & 26S Proteasome [Ubiquitin-Vinyl Sulfone (Ub-VS) treated] (UBI-64-1010-096), 5x DUB Assay Buffer (UBI-64-2001-500), 4x Ubiquitin-Rhodamine 110 substrate (UBI-60-0122-500) and 5x DUB Assay Stop Buffer (UBI-64-2002-500). The 26S proteasome product in this kit was prepared using the same protocol as described in Wang et al. (2007) where DUB activity was removed through washing and treatment with Ub–VS which forms an adduct with the active site cysteine in DUBs of the thiol protease class (Lee et al., 2010). This kit may be of interest for screening inhibitors of ‘proteasome-activated’-USP14.

Deconjugating enzymes (DCEs) are proteases that reverse the modification of proteins by a single ubiquitin or ubiquitin-like protein (UBL) and remodel polyubiquitin (or poly-UBL) chains on target proteins (Reyes-Turcu et al., 2009). DUBs represent the largest family of DCEs and regulate ubiquitin dependent signaling pathways. DUB activities include the generation of free ubiquitin from precursor molecules, the recycling of ubiquitin following substrate degradation to maintain cellular ubiquitin homeostasis and the removal of ubiquitin or UBL modifications through chain editing to rescue proteins from proteasomal degradation or to influence cell signaling events (Komander et al., 2009). There are two main classes of DUB enzymes: cysteine proteases and metalloproteases. Ubiquitin specific protease 14 (USP14) is a member of the cysteine protease enzyme family and cloning of the gene was first described by Deshpande et al. (1996).

The ubiquitin proteasome system (UPS) targets selected proteins for degradation by the 26S proteasome. The initial steps in this pathway generate proteins that are covalently tagged with a polyubiquitin chain that is then recognized by ubiquitin receptors of the 26S proteasome. This is a large complex composed of a 20S catalytic core particle and two 19S regulatory particles (Kok et al., 1993) that catalyse the final step in the path- way. While the 20S particle is composed of a catalytic chamber for protein degradation, collectively the proteins that comprise the 19S particle perform several proteasomal functions that include recognition of ubiquitylated substrates, cleavage of the polyubiquitin chain for ubiquitin recycling, control of access to the 20S proteolytic chamber, and substrate unfolding and subsequent translocation into the 20S core particle for degradation (Boehringer et al., 2012). Mammalian proteasomes are associated with three DUBs: USP14, UCHL5 (UCH37) and RPN11 (POH1). USP14 and UCHL5 reside on the regulatory particle and remove ubiquitin from the substrate before substrate degradation whereas RPN11 activity is delayed until the proteasome is committed to de- grading the substrate (Lee et al., 2010). The DUB activity of USP14 is known to be activated through its interaction with the proteasome complex.

Specifications


• Species:                       human

• Source:                        USP14 = E. coli; 26S Proteasome = transformed HEK-293 cells

• Quantity:                     96 assay tests

• Formulation:               DTT-containing buffer

• Molecular Weight:      USP14 = ~58.5 kDa; 26S Proteasome = ~2500 kDa

• Stability/Storage:        12 months at -70°C; Avoid multiple freeze/thaw cycles


Kit components (also available individually)