cDNA cloning of a human 100 kDa de‐ubiquitinating enzyme: the 100 kDa human de‐ubiquitinase belongs to the ubiquitin C‐terminal hydrolase family 2 (UCH2)

Abstract

The full length cDNA encoding a 100 kDa human de-ubiquitinating enzyme, referred to as de-ubiqnitinase was obtained using one clone selected from a randomly sequenced human brain cDNA library and specific primers. The sequence of 18 peptides generated from the de-ubiquitinase isolated from outdated human erythrocytes matched perfectly with the predicted amino acid sequence, which would encode a protein containing 858 amino acids (calculated M r = 95,743 Da). Homology search disclosed that the protein is a member of a large family of ubiquitin C-terminal hydrolases (UCH2), that was defined on the basis of the presence of two specific patterns, 'the Cys-and His-domains', which are likely to be involved in the de-ubiquitinating activity [7]. An additional conserved region, 'the aspartic acid domain', was also identified, the functional role of which is unknown.

The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

“…on May 10, 2018 by guest http://mcb.asm.org/ nucleophile (Cys), a general base (His), and an acidic residue (Asp) (4,23,107). In Rpn11, a number of conserved aspartates and histidines are present downstream of the conserved Cys box and could potentially serve these functions.…”

Identification, Functional Characterization, and Chromosomal Localization of USP15, a Novel Human Ubiquitin-Specific Protease Related to the UNP Oncoprotein, and a Systematic Nomenclature for Human Ubiquitin-Specific Proteases

“…E-mail: Rohan.Baker@anu.edu.au. addition, proteins from other eukaryotes that contain these conserved sequence motifs have also been demonstrated to have DUB activity (e.g., Papa and Hochstrasser, 1993;Falquet et al, 1995;Huang et al, 1995;Wilkinson et al, 1995;Gilchrist et al, 1997;Everett et al, 1997;Zhu et al, 1996Zhu et al, , 1997Naviglio et al, 1998;Baek et al, 1998;Sloper-Mould et al, 1999), indicating that the presence of the Cys and His boxes strongly correlates with DUB activity. At least 40 Ubps from nonyeast (S. cerevisiae and Schizosaccharomyces pombe) eukaryotes have been reported, with at least 14 of these in humans (Wilkinson, 1997;Baek et al, 1997Baek et al, , 1998Fujiwara et al, 1998;Timms et al, 1998;SloperMould et al, 1999) and many more examples in expressed-sequence tag (EST) databases.…”

The measurement of ubiquitin and ubiquitinated proteins

“…Although the large number and structural diversity of deubiquitinating enzymes has precluded identification of specific inhibitors, the fact that all known members of this family are exquisitely dependent on active site cysteine residues, makes thiol-blocking reagents attractive candidates as general inhibitors. In fact, N-ethylmaleimide (NEM) has become the inhibitor of choice in this regard [91]. In experiments where it is important to preserve polyubiquitinated protein conjugates that are formed in vivo, NEM is added to the cell lysis buffer at a final concentration of 10± 25 mM.…”