Divergent N-terminal sequences of a deubiquitinating enzyme modulate substrate specificity.
Ubiquitin-specific processing proteases (UBPs) are characterized by a conserved core domain with surrounding divergent sequences, particularly at the N-terminal end. We previously cloned two isoforms of a testis UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini that target the two isoforms to different subcellular locations (Lin, H., Keriel, A., Morales, C. R., Bedard, N., Zhao, Q., Hingamp, P., Lefrancois, S., Combaret, L., and Wing, S. S. (2000) Mol. Cell. Biol. 20, 6568-6578). To determine whether the N termini also influence the biochemical functions of the UBP, we expressed UBP-t1, UBP-t2, and the common core domain, UBP core, in Escherichia coli. The three isoforms cleaved branched triubiquitin at >20-fold faster rates than linear diubiquitin, suggesting that UBP-testis functions as an isopeptidase. Both N-terminal extensions inhibited the ability of UBP-core to generate free ubiquitin when linked in a peptide bond with itself, another peptide, or to small adducts. The N-terminal extension of UBP-t2 increased the ability of UBP-core to cleave branched triubiquitin. UBP-core removed ubiquitin from testis ubiquitinated proteins more rapidly than UBP-t2 and UBP-t1. Thus, UBP enzymes appear to contain a catalytic core domain, the activities and specificities of which can be modulated by N-terminal extensions. These divergent N termini can alter localization and confer multiple functions to the various members of the large UBP family.
Polypeptide Laboratory, Department of Medicine and Department of Biochemistry, McGill University, Montreal, Quebec, H3A 2B2, Canada.
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D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013737:Testis / Q000378:metabolism; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000378:metabolism*
D009124:Muscle Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014452:Ubiquitins; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase; C464656:Usp2 protein, rat