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 pathway. 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). UCHL5 and USP14 reside on the regulatory particle and remove ubiquitin from the substrate before substrate degradation whereas RPN11’s activity is delayed until the proteasome is committed to degrading the substrate (Lee, et al., 2010). The DUB activity of USP14 is known to be activated by proteasomes. The 26S proteasome preparation in this product was prepared using the same protocol as described in Wang et al. (2007). The 26S proteasome-associated DUB activity was removed through washing and treatment with ubiquitin– vinylsulphone (Ub–VS) which forms an adduct with the active site cysteine in DUBs of the thiol protease class (Lee, et al., 2010).

References:

Boehringer, J et al. (2012) Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation, Biochemical J 448, 55-65.

Kok, K et al. (1993) A gene in the chromosomal region 3p21 with greatly reduced expression in lung cancer is similar to the gene for ubiquitin-activating enzyme, Proc Natl Acad Sci USA 90, 6071- 6075.

Lee, BH et al. (2010) Enhancement of proteasome activity by a small-molecule inhibitor of USP14, Nature 467, 179-184.

Wang, X. et al. (2007) Mass spectrometric characterization of the affinity-purified human 26S proteasome complex, Biochemistry 46, 3553-3565.

Ubiquigent Limited is a specialist developer and supplier of Drug Discovery Services, high quality Research Tools and Chemistry to the life science research community worldwide.

Ubiquigent’s scientific and business interests have a clear focus; namely the ubiquitin, ubiquitin-like, and integrated signalling systems.

The Company has established its scientific and business credentials with both academic researchers undertaking fundamental scientific discovery and pharmaceutical and biotechnology company scientists exploring the potential of ubiquitin cascade-focused drug discovery.

Ubiquigent benefits from high calibre backing including from private US investors, the UK Medical Research Council, and the University of Dundee. The Company’s headquarters and laboratory operations are based in the UK and are located in a state-of-the-art facility adjacent to the MRC Protein Phosphorylation and Ubiquitylation Unit at the University of Dundee (both founded by Professor Sir Philip Cohen). In addition to Ubiquigent’s own facilities and capabilities, such proximity provides ready access to a huge range of additional scientific expertise (1,000+ life-science researchers on site), technological competencies, and assay and analytical platforms.

Ubiquigent staff have a wealth of scientific experience, technological competencies and commercial expertise, gained from within academia, the life science research reagents and services sector, and the pharmaceutical industry, that enables the Company to offer a level of understanding and service to its customers and clients worldwide that few companies may match.