title: A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond.
abstract: Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin. The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin. The single exception is when proline is the amino acid immediately following ubiquitin; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases. We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes. N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction. The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally. However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins. Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner.
authors.affiliation: Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia.
authors.forename: C A
authors.initials: CA
authors.lastname: Gilchrist
authors.forename: D A
authors.initials: DA
authors.lastname: Gray
authors.forename: R T
authors.initials: RT
authors.lastname: Baker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins; D011392:Proline / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D017386:Sequence Homology, Amino Acid; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; C486203:USP4 protein, human; D014452:Ubiquitins; C082325:Usp4 protein, mouse; D011392:Proline; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases