Ubiquitin C-terminal hydrolase 1 (UCHL1) has several other names, such as ubiquitin carboxyl esterase L1, ubiquitin thiolesterase, neuron-specific protein PGP9.5 and Park5. It was originally identified as a major component of the neuronal cytoplasm from 2-dimensional gel analysis of brain tissues, and was given the name PGP9.5 (1). The protein is extremely abundant, and was estimated to be present at a concentration of 200-500 µg/g wet weight, representing a major protein component of neuronal cytoplasm (1). This is ~1-2% of total brain protein (2).
Ubiquitin C-terminal hydrolase enzyme activity resided in the PGP9.5 protein, resulting in the name ubiquitin C-terminal hydrolase 1 (2). The name Park5 derives from the fact that a very rare form of familial Parkinson’s disease is associated with an I93M mutation in the UCHL1 gene, which reduces the ubiquitin hydrolase activity. A common variant of UCHL1, called S18Y, is protective against Parkinson’s disease. This is the first of a family of ubiquitin C-terminal hydrolases, and is expressed heavily in brain and testis. The ubiquitin C-terminal hydrolases cleave ubiquitin from other molecules. This activity is important to generate mono-ubiquitin from genes which encode ubiquitin, most of which actually generate multiple tandemly repeated ubiquitin units. The activity is also important to remove ubiquitin from partially degraded proteins, allowing the ubiquitin monomer to be recycled. Regulation of the ubiquitin pathway is very important and many disease states are associated with defects in this pathway. The covalent ubiquitin conjugates may then be degraded in the proteasome. Point mutations in the UCHL1 gene are associated with forms of human Parkinson’s disease (3). Recent studies suggest that UCHL1 also has a ubiqutinyl ligase activity, being able to couple ubiquitin monomers by linking the C-terminus of one with lysine 63 of the other (3). Since UCHL1 is heavily expressed in neurons, antibodies to UCHL1 can be used to identify neurons in histological sections and in tissue culture.
His-tagged-UCHL1, was expressed and purified from E. coli BL21 using immobilized metal affinity chromatography. Protein concentration was determined using BSA Protein Bradford, and confirmed with a densitometry-based ImagJ quantification using BSA as standard. Picture shows the Coomassie gel of UCHL1 (Lot #3152013) and BSA at serial dilution. UCHL1 runs at ~37 kDa.
A cDNA encoding full length human UCHL1 was inserted into a eukaryotic expression vector with a C-terminal His-Tag and subsequently transformed into E. coli BL21 cells. Cells were grown in LB medium supplemented with ampicillin at 37°C. Cells were harvested after induced protein expression for 6 hrs. Soluble protein was purified using immobilized metal affinity chromatography. Yields of soluble UCHL1 protein were estimated by performing SDS-PAGE and staining with Coomassie Blue R250. Purified protein was concentrated following extensive dialysis in PBS to 1 mg/mL, and stored at -70°C (recommended for long term storage). For the subsequent analyses, the concentration of UCHL1 was obtained using the Bradford method and confirmed with a densitometry-based ImageJ quantification.
1. Doran JF, Jackson P, Kynoch PA, Thompson RJ. Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis. J Neurochem. 40:1542-7 (1983).
2. Wilkinson KD, Lee KM, Deshpande S, Duerksen-Hughes P, Boss JM, Pohl J. The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science. 1989 246:670-3 (1989).
3. Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT Jr. The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson’s disease susceptibility. Cell 111:209-18 (2002).
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