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NameCatalogue NumberSizePriceAdd to Basket
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Catalogue Number:64-0040-050Size:50 µgPrice:£325Add To Basket
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Specieshuman
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SourceInsect sf21
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Quantity50 µg
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Storage-70°C
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Concentration0.5 mg/ml
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Formulation50 mM HEPES pH 7.5, 150 mM sodium chloride, 2 mM dithiothreitol, 10% glycerol
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Molecular WeightUSP1 = 90.5 kDa/UAF1 = 76.2 kDa
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Stability12 months at -70°C; Ubiquigent strongly recommends aliquoting for single use as required
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Protein SequenceAccession number: USP1 = NP_003359 and UAF1 = NP_065890. For full protein sequence information download the Certificate of Analysis pdf.
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QA; Protein IdentificationConfirmed by mass spectrometry.
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QA; ActivityDeubiquitylase Enzyme Assay: The activity of 6His-USP1/UAF1 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 6His-USP1/UAF1 was compared confirming the deubiquitylating activity of 6His-USP1/UAF1.
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 1 (USP1) is a member of the cysteine protease enzyme family and cloning of the human gene was first described by Fujiwara et al. (1998). USP1 has been shown to act as a negative regulator of the Fanconi anaemia pathway. Inhibition of USP1 by siRNA knockdown increased the monoubiquitylation of the Fanconi anaemia effector protein, FANCD2, and increased cellular resistance to DNA cross-linking agents, suggesting that USP1 deubiquitylates FANCD2 (Huang et al., 2006). USP1 associated factor 1 (UAF1) not only interacts with and stabilizes USP1 but also stimulates its enzymatic activity. UAF1 binding increases the catalytic turnover (Kcat) but does not increase the affinity of the USP1 enzyme for the substrate (KM) (Cohn et al., 2007). To explore USP1 autocleavage, a USP1 variant was expressed where the amino acids on positions 670 and 671 were changed from Gly-Gly to Ala-Ala. This mutation inhibited both the cleavage and degradation of USP1 (Huang et al., 2006). USP1 plays important roles in cancer-related processes, such as the DNA damage response, and the maintenance of the undifferentiated state of osteosarcoma cells. Inhibiting the function of the USP1/UAF1 complex sensitizes cancer cells to chemotherapy, suggesting that this complex is a relevant anticancer target (Garcia-Santisteban et al., 2012).