USP21 CD(196-565) [GST-tagged]


Catalogue Number
64-0037-050
Product Size
50 µg
Price £
£325
Accession Number
NP_001014443
Residues Expressed
196-565
Certificate of Analysis Size
50 µg
Species
human
Source
E.coli
Quantity
50 µg
Storage
-70°C
Concentration
0.5 mg/ml
Formulation
50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
Molecular Weight
67.5
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_001014443. For full protein sequence information download the Certificate of Analysis pdf.
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

Deubiquitylase Enzyme Assay: The activity of GST-USP21 was validated by determining the increase in fluorescence measured as a result of the enzyme catalysed cleavage of the fluorogenic substrate Ubiquitin-Rhodamine110-Glycine generating Ubiquitin and Rhodamine110-Glycine. Incubation of the substrate in the presence or absence of GST-USP21 was compared confirming the deubiquitylating activity of GST-USP21.


Background

Deconjugating enzymes (DCEs) are proteases that process ubiquitin or ubiquitin-like gene products, 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). The deubiquitylating - or deubiquitinating - enzymes (DUBs) represent the largest family of DCEs and regulate ubiquitin dependent signalling pathways. The activities of the DUBs 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 ubiquitin-like proteins (UBL) modifications through chain editing to rescue proteins from proteasomal degradation or to influence cell signalling events (Komander et al., 2009). There are two main classes of DUB, cysteine proteases and metalloproteases. Ubiquitin specific protease 21 (USP21) is a member of the cysteine protease enzyme family and cloning of the gene was first described by Gong et al. (2000). USP21 cleaves ubiquitin polymers, and with reduced activity also targets the UBL ISG15 but not NEDD8 (Ye et al., 2011). USP21 has been shown to be involved in the regulation of transcriptional initiation through the deubiquitylation of histone H2A as well as playing a role in the regulation of tumour necrosis factor α (TNFα) induced nuclear factor κβ (NF-κβ) activation by deubiquitylating receptor-interacting protein 1 (RIP1) (Nakagawa et al., 2008; Xu et al., 2010). Proteomic analyses also identified microtubule affinity-regulating (MARK) protein kinases and phosphatases as USP21 interactors, suggesting roles for USP21 in cell signalling (Li et al., 2005). In a recent screen of 66 DUBs tagged with green fluorescent protein (GFP), USP21 was found to be unique by showing clear association with both centrosomes and microtubules (Urbe et al., 2012).


References

Gong L, Kamitani T, Millas S and Yeh ET (2000) Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins. The Journal of biological chemistry, 275, 14212-14216.

Komander D, Clague MJ and Urbe S (2009) Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol, 10, 550-563.

Li Z, Wang D, Messing EM and Wu G (2005) VHL protein-interacting deubiquitinating enzyme 2 deubiquitinates and stabilizes HIF-1alpha. EMBO reports, 6, 373-378.

Nakagawa T, Kajitani T, Togo S, Masuko N, Ohdan H, Hishikawa Y, et al. (2008) Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation. Genes & development, 22, 37-49.

Reyes-Turcu FE, Ventii KH and Wilkinson KD (2009) Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annual review of biochemistry, 78, 363-397.

Urbe S, Liu H, Hayes SD, Heride C, Rigden DJ and Clague MJ (2012) Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions. Molecular biology of the cell, 23, 1095-1103.

Xu G, Tan X, Wang H, Sun W, Shi Y, Burlingame S, et al. (2010) Ubiquitin-specific peptidase 21 inhibits tumor necrosis factor alpha-induced nuclear factor kappaB activation via binding to and deubiquitinating receptor-interacting protein 1. The Journal of biological chemistry, 285, 969-978.

Ye Y, Akutsu M, Reyes-Turcu F, Enchev RI, Wilkinson KD and Komander D (2011) Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21. EMBO reports, 12, 350-357.