A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14

Abstract

A C-terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site-directed probe that irreversibly modifies a subset of Ub C-terminal hydrolases (UCHs) and Ub-specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [(125)I]UbVS labeling with N-ethylmaleimide and Ub aldehyde, but also by genetic analysis. [(125)I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [(125)I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S-associated USP14 with [(125)I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.

Fig. 1. Synthesis and characterization of UbVS. (A) C-terminal modification of ubiquitin 1 to generate ubiquitin vinyl sulfone 6. A 25 mg aliquot of 1 was converted to ubiquitin₇₅-ethyl ester 2 by treatment with 2.5 mg of trypsin in the presence of 2.5 M glycine ethyl ester (GlyOEt) and 20% PEG 20 000. Ubiquitin₇₅-ethyl ester was treated with hydrazine monohydrate and HCl to generate ubiquitin₇₅-hydrazine 3 which was dialyzed against water and converted to ubiquitin₇₅-azide 4 by treatment with 0.5 M nitrous acid for 1 min at –5°C. Ubiquitin₇₅-azide immediately reacted with the TFA salt of glycine vinyl sulfone 5 in the presence of TEA, generating 6. (B) Purified UbVS is resolved on an analytical C4 column using a 0.1% formic acid/acetonitrile buffer system. Eluate was analyzed by on-line ES-MS. The indicated multicharged species correspond to a mol. wt of 8624.9 Da, in agreement with the predicted molecular weight of UbVS (8625 Da). (C) Recombinant, purified UCH-L3 was incubated with a substochiometric amount of [¹²⁵I]UbVS in PBS for 45 min at 37°C with or without pre-treatment with 2 mM N-ethylmaleimide. Protein conjugates were resolved by 12.5% SDS–PAGE under reducing conditions, and visualized by silver stain (left panel) and autoradiography (right panel).

Fig. 2. [¹²⁵I]UbVS specifically labels a subset of yeast DUBs. (A) A 100 µg aliquot of post-nuclear lysates from DUB deletion strains was incubated with 1 × 10⁶ c.p.m. of [¹²⁵I]UbVS for 45 min at 37°C. Reactions were quenched with sample buffer, resolved by 10% SDS–PAGE and analyzed by autoradiography. The identity of the bands was assigned based on the absence of a band corresponding to the molecular weight of a DUB deleted in that strain. (B) Lysates from wild-type yeast were pre-incubated with increasing concentrations of Ubal competitor for 30 min at room temperature prior to addition of [¹²⁵I]UbVS as described in (A).

Fig. 3. [¹²⁵I]UbVS labels mammalian DUBs. (A) Single cell suspensions were prepared from tissues of a male B6 mouse: muscle (MU), brain (BR), kidney (KI), thymus (TH) and spleen (SP). A 50 µg aliquot of lysate was treated with 1 × 10⁶ c.p.m. [¹²⁵I]UbVS as described in Figure 2A and resolved by 10% SDS–PAGE. (B) A 20 µg aliquot of post-nuclear lysates from NIH 3T3 cells was pre-treated with increasing concentrations of Ubal as competitor for 30 min at 37°C followed by labeling with [¹²⁵I]UbVS. (C) A 50 µg aliquot of lysates from EL-4 cells was treated with 2 µM UbVS at 37°C for 1 h, reactions were resolved by 10% SDS–PAGE and immunoblotted with the r201 rabbit antiserum against USP7.

Fig. 4. USP14 associates with the 26S proteasome. (A) EL-4 cell lysates were fractionated on a Superose 6 FPLC column to isolate high molecular weight complexes, as described in Materials and methods. Fractions were labeled with 0.5 × 10⁶ c.p.m. of [¹²⁵I]UbVS and resolved by SDS–PAGE. (B) A 80 µg aliquot of EL-4 cell lysates was treated with 2 × 10⁶ c.p.m. of [¹²⁵I]UbVS and immunoprecipitated with anti-20S proteasome antiserum using proteasome IP buffer or denatured with 1% SDS and immunoprecipitated with anti-USP14 antiserum HM433 using NET buffer. (C) EL-4 lysates were fractionated on a Superose 6 column as described in (A). A 1 ml aliquot of each fraction was incubated with [¹²⁵I]UbVS and immuno precipitated for the proteasome as in (B) (top panel). A 50 µl aliquot of each fraction was analyzed for the presence of proteasome subunits by immunoblot with the indicated antibodies against components of the 20S and 19S complexes (lower panels).

Fig. 5. [¹²⁵I]UbVS labeling of proteasome-associated USP14 is increased upon proteasome inhibition. (A) A total of 5 × 10⁶ EL-4 cells were treated with 50 µM NLVS for the indicated times. Lysates were normalized for total protein and incubated with [¹²⁵I]UbVS or [¹²⁵I]NLVS for 1 h. Proteasomes were immunoprecipitated as described in Figure 4B. (B) Intensities of USP14 bands were quantified by densitometry. (C) EL-4 cells were incubated with 50 µM NLVS or ZL₃VS or 4 µM epoxomicin for the indicated times. Proteasomes were immunoprecipitated as described in Figure 4B.

Fig. 6. Activity of USP14 is increased in response to proteasome inhibition. (A) Subcellular fractions from EL-4 cells previously treated with 50 µM NLVS for 5 h were incubated with [¹²⁵I]UbVS, resolved by 10% SDS–PAGE and visualized by autoradiography. 1hS, 1 h supernatant; 5hS, 5 h supernatant; 5hP, 5 h pellet. (B) Parallel samples were resolved by 10% SDS–PAGE and immunoblotted with anti-USP14 and anti-Mss1 antisera. (C) Subcellular fractions prepared from EL-4 cell extracts were incubated with 50 µM NLVS for 3 h, resolved by 10% SDS–PAGE and visualized by autoradiography (upper panel), and by immunoblot using anti-USP14 (lower panel). The intensities of USP14 bands obtained from four independent experiments were quantified by densitometry and normalized to the amount of USP14 labeled by [¹²⁵I]UbVS (upper panel) and USP14 protein (lower panel) observed in 1 h supernatant fractions.