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Functions of Ubiquitin Specific Proteases Essay

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Eukaryotic cells rely on the ubiquitination of proteins for the proper control of their internal processes. Adding multiple ubiquitin groups aids in the breakdown of proteins, whereas adding only one ubiquitin serves regulatory roles other than degradation. The enzymatic activity of two protease classes frees ubiquitin groups from associated proteins: Ubiquitin specific proteases (USP) and Ubiquitin C-terminal hydrolases (UCH). USPs are strongly involved in eukaryotic cellular functions and are found in copious amounts. Certain protein ligases attach ubiquitin to proteins, which degrades them and interferes with normal cellular functions. The discovery of a specific de-ubiquitinating enzyme (HAUSP/USP7) found in humans was due to its…show more content…
The labels associated with the resulting proteins from previous steps were rinsed and removed. USP7 was purified and utilized in sedimentation trials in varying concentrations. Absorbance analysis was carried out after the samples were centrifuged. EBNA1 was purified, ubiquitinated, and combined with USP7, which was purified in previous steps. SDS-PAGE was used to disrupt the protein interaction and separate them. The separated fragments were then used in a western blot, and analyzed after probing. The affinity of USP7 for EBNA1 and p53 was tested using a stepwise combination of titration, incubation, and fluorescence analysis. Lastly, amino acid residues of EBNA1and USP7 were studied by gel filtration and size-exclusion chromatography. USP7 was found as hexahistidine fusion after being inserted (through a vector) in insect cells. The de-ubiquitinating capacity of USP7 for conjugated ubiquitin groups was observed on EBNA1, and was shown to an efficient process. The capacity of USP7 to de-ubiquitinate specific proteins in various environments (acidity, presence of inhibitors, and salt concentration) was studied. As seen in figure 2, USP7 sensitivity to salt concentration and acidity showed a steep slope followed by a plateau at lower salt concentrations and higher pH levels, while lower pH and higher salt concentrations rendered a steeper, less plateau-like graph. USP7 was found to be a monomer with multiple domains, each involved
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