Zinc Is Required for the Catalytic Activity of the Human Deubiquitinating Isopeptidase T

Abstract

Two recombinant human isopeptidase T isoforms, ISOT-S and ISOT-L, differing by an insertion of 23 amino acids in ISOT-L, were previously classified as thiol proteases. Both contain one Zn2+-binding site of high-affinity, which is part of a cryptic nitrilo-triacetate-resistant pocket (site 1). A second Zn2+ site (site 2) was disclosed when both isoforms of the holoenzyme were incubated with an excess of Zn2+. The firmly bound Zn2+ of site 1 could be removed either slowly by dialysis against 1,10-phenanthroline at pH 5.5 or rapidly by treatment at pH 3.0 in the presence of 6 M urea followed by gel filtration at neutral pH. Zn2+ in site 1, but not in site 2, is essential for proteolytic activity because apoproteins were inactive. Inhibition of the catalytic activity was not due to a loss of ubiquitin binding capacity. CD spectra of both isoforms disclosed no major structural differences between the apo- and holoenzymes. The reconstitution of apoenzyme with Zn2+ under nondenaturing conditions at pH 5.5 completely restored enzymatic activity, which was indistinguishable from the reconstitution carried out in urea at pH 3.0. Thus, both human ISOTs are either thiol proteases with a local structural Zn2+ or monozinc metalloproteases that might use in catalysis a Zn2+-activated hydroxide ion polarized by Cys335.

Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity

“…The exact mechanism by which (PyDT) 2 Zn increases the proteasomal chymotrypsin-like activity within the cell is not understood and needs to be further investigated. It has also been reported that human deubiquitinating isopeptidase T has three sets of zinc-binding sites and that zinc binding to high affinity sites is required for its activity (Gabriel et al, 2002). However, by binding to the set of low affinity sites, zinc ions completely abolish isopeptidase T enzymatic activity.…”

“…4A). Since zinc is required for the catalytic activity of the human deubiquitinating isopeptidase T (Gabriel et al, 2002), it is possible that isopeptidase activity located on 19S regulatory subunit of the 26S proteasome could also be activated by (PyDT) 2 Zn or zinc ions released from the complex during the possible intracellular degradation. The fact that the high level of ubiquitinated proteins still remained until the end of treatment could be explained by the proteasomal inhibition observed again at the end of the treatment and by the formation of aggresomes.…”

Domain Analysis Reveals That a Deubiquitinating Enzyme USP13 Performs Non-Activating Catalysis for Lys63-Linked Polyubiquitin

“…The tandem UBA domains associate with the distal Ub units when USP5 catalyzes the hydrolysis of polyUb chains [18]. ZnF is the activation domain of USP5 that is recognized by the C-terminal extension of Ub [19], [20]. The catalytic domain consists of the C-box and H-box lobes including substrate binding sites.…”

Recognition of Polyubiquitin Isoforms by the Multiple Ubiquitin Binding Modules of Isopeptidase T

“…IsoT-3 is encoded by a different gene, USP13, and shares 54.8% identity with IsoT-S (31). Whereas most in vitro studies aimed at understanding the substrate specificity of IsoT have been conducted using human IsoT, very little is known about the substrate specificity of the other two human isoforms (28,(33)(34)(35)(36). IsoT disassembles at least four types of polyubiquitin isoforms: K48-, K63-, K29/K6-linked, and linear chains (26,28).…”