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)

Falquet, Laurent; Paquet, Nicole; Frutiger, Séverine; Hughes, Graham J.; Hoang-Van, Khan; Jaton, Jean‐Claude

doi:10.1016/0014-5793(95)01287-7

Cited by 22 publications

(26 citation statements)

References 25 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

Glickman

Rubin

Fried

et al. 1998

Molecular and Cellular Biology

473

433

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The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.In eukaryotes, the elimination of many short-lived proteins requires their covalent attachment to ubiquitin (43). This pathway is involved in a wide variety of regulatory mechanisms, with substrates including cyclins and CDK inhibitors (49), membrane proteins such as CFTR (104), p53 (84), NF-B (71), c-Fos, c-Jun, and luminal components of the endoplasmic reticulum (9). Multiubiquitin chains target proteins for degradation by the proteasome, an ϳ2-MDa proteolytic complex (reviewed in references 12, 55, 61, and 80).The mechanism of the proteasome is thought to involve unfolding of a protein substrate and translocation from one subcompartment of the enzyme to another prior to degradation. This model is based primarily on the crystal structure of the proteasomal core particle (CP). The CP has a barrel-like shape, with the proteolytic active sites facing the inner chamber, or lumen. In the proteasomal CP from Thermoplasma acidophilum, openings into the lumen are found only at the ends of the barrel (59) and are therefore thought to function as channels for the proteolytic substrate. Because these channels are narrow, it is likely that proteolytic substrates must be unfolded prior to entry into the lumen of the CP. Bas...

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Section: Resultsmentioning

confidence: 99%

The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

Glickman

Rubin

Fried

et al. 1998

Molecular and Cellular Biology

473

433

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“…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.…”

Section: Introductionmentioning

confidence: 94%

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

et al. 1999

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“…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.…”

Section: Pitfalls To Avoid and Other Helpful Suggestions 291 Enzymementioning

confidence: 99%