OTUD3 [GST-tagged]


Catalogue Number
64-0035-050
Product Size
50 µg
Price £
£430
Accession Number
NP_056022
Residues Expressed
1-629
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
~72 kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: NP_056022. 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-OTUD3 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-OTUD3 was compared confirming the deubiquitylating activity of GST-OTUD3.


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. OTUD3 is a cysteine protease and is a member of the OTU superfamily of proteins (Balakirev et al., 2003). Cloning of the human gene was first described by Seki et al. (1999). OTU enzymes play important roles as negative-feedback regulators in NF-κB signalling, interferon signalling and in p97 (cdc48)-mediated processes although the cellular functions of most OTU enzymes remain to be discovered. Ovarian tumour family DUBs contain a papain-like catalytic core of ~180 amino acids. In addition to their catalytic domain, many OUT members have additional ubiquitin-binding domains (UBDs). At least 20 different UBD families have been described, and knowledge of linkage-specific UBDs have provided the means to understand the roles of different ubiquitin linkages in cells (Licchesiet al., 2012).


References

Balakirev MY, Tcherniuk SO, Jaquinod M and Chroboczek J (2003) Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep 4, 517-522.

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.

Licchesi JD, Mieszczanek J, Mevissen TE, Rutherford TJ, Akutsu M, Virdee S, et al. (2012) An ankyrin-repeat ubiquitin-binding domain determines TRABID's specificity for atypical ubiquitin chains. Nature Structural & Molecular Biology 19, 62-71.

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

Seki N, Hattori A, Hayashi A, Kozuma S, Sasaki M, Suzuki Y, etal. (1999) Cloning and expression profile of mouse and human genes, Rnf11/RNF11, encoding a novel RING-H2 finger protein. Biochim Biophys Acta 1489, 421-427.