Fig 3. ETPs inhibit JAMM domain proteases.

(A) ETPs inhibit Rpn11 activity by binding zinc. The IC₅₀ for inhibition of Rpn11 activity by SOP6 was determined in the presence and absence of a Zn(cyclen)²⁺ coordination complex. The Ub4-peptide substrate was used for this assay as depicted. Error bars represent s.d., n = 4 wells, from 1 representative of 3 independent experiments. (B) Purified Rpn11•Rpn8 was incubated with K48-linked diubiquitin in the presence of 40 μM or 10 μM of the indicated compounds, and reactions were fractionated by SDS–PAGE and visualized with Coomassie blue. (C) Zn²⁺ counteracts gliotoxin induced accumulation of poly-ubiquitinated species. HCT116 cells were treated for 3 hours with 10 μM gliotoxin or CZM in the absence or presence of 150 μM ZnSO4 in the culture medium, and cell lysates were fractionated by SDS–PAGE and immunoblotted with antibodies against ubiquitin. See also Table S2.