An Arsenite Relay between PSMD14 and AIRAP Enables Revival of Proteasomal DUB Activity

Abstract

Maintaining 26S proteasome activity under diverse physiological conditions is a fundamental requirement in order to maintain cellular proteostasis. Several quantitative and qualitative mechanisms have evolved to ensure that ubiquitin-proteasome system (UPS) substrates do not accumulate and lead to promiscuous protein-protein interactions that, in turn, lead to cellular malfunction. In this report, we demonstrate that Arsenite Inducible Regulatory Particle-Associate Protein (AIRAP), previously reported as a proteasomal adaptor required for maintaining proteasomal flux during arsenite exposure, can directly bind arsenite molecules. We further show that arsenite inhibits Psmd14/Rpn11 metalloprotease deubiquitination activity by substituting zinc binding to the MPN/JAMM domain. The proteasomal adaptor AIRAP is able to directly relieve PSMD14/Rpn11 inhibition. A possible metal relay between arsenylated PSMD14/Rpn11 and AIRAP may serve as a cellular mechanism that senses proteasomal inhibition to restore Psmd14/Rpn11 activity.

Keywords: AIRAP; PSMD14/RPN11; arsenite; proteasome; protein misfolding; proteostasis.

Figure 1

Recombinant AIRAP binding to a PAO matrix. (A) Recombinant mouse AIRAP was expressed as a C-terminus 6×His fusion protein and Ni-NTA affinity purified. Purified material was incubated with a control (NH₂) or amino-phenylarsine (PAO) immobilized Sepharose 4B matrix in the presence of the cysteine alkylating N-ethylmaleamide (NEM), denaturing conditions (6M guanidine–HCl), reducing conditions (THP), sodium arsenite (As) or soluble amino-phenylarsine (PAO). Immobilized material was extensively washed, eluted by boiling with 2% SDS and subjected to SDS-PAGE and subsequent immunobloting to evaluate AIRAP content. (B) The impact of metals on AIRAP binding to a PAO matrix was performed as in 1A by performing the binding in the presence of the indicated metals. The arrow indicates the full-length recombinant product of AIRAP.

Figure 2

Dependence on zinc and MST measured affinity. (A) The presence of arsenite bound to AIRAP was determined by ICP-OES. Recombinant AIRAP was incubated in the presence (Zn⁺) or absence (Zn⁻) of zinc, prior to arsenite exposure. Bound material was extensively washed and arsenite content determined. (B) Recombinant AIRAP (1 μM) was incubated with a range of arsenite dilutions as indicated, and intrinsic fluorescence was measured by MST. Analysis revealed Kd values of 7 nM ± 0.87 nM.

Figure 3

Endogenous proteasomal AIRAP binds arsenite. Cell lysates acquired from arsenite-treated cells were incubated with a PAO matrix. AIRAP and 20S proteasomal content (PSMA1) were evaluated by immunoblot (lanes 1–3). Alternatively, PAO matrix binding was performed using soluble proteasomes purified from arsenite-treated cells (lanes 4–6). Where indicated, PAO matrix was washed with high salt to evaluate impact on PAO binding (lanes 3 and 6).

Figure 4

PSMD14 binding to PAO. Soluble proteasomes were incubated with PAO matrix, extensively washed and PSMD14, 19S (S5a), and 20S (PSMA1) content revealed by immunoblot.

Figure 5

MPN domain mediates PSMD14 binding to PAO. PSMD14 WT or MPN mutant was evaluated towards its ability to bind a PAO matrix. Proteasomes were purified from cells expressing GFP (serving as a control), PSMD14-GFP (WT) or PSMD14-Flag (MPN mutant). Proteasomal content (input) reveals no requirement for a functional MPN domain for proteasomal binding (lanes 3 and 5). However, PSMD14 arsenite binding (PAO matrix) is detected only in the PSMD14 WT and not MPN mutant (lanes 4 vs. 6).

Figure 6

Proteasomal PSMD14 activity assay. (A) 19SΔUU proteasomes containing the PSMD14/Rpn11 DUB do not contain Usp14/Ubp6 or Uch37, while WT proteasomes contain all three DUBs. (B) Polyubiquitinated Sic1 was mixed with 19SΔUU proteasomes and samples were retrieved at the indicated time points. Proteasomal PSMD14, AIRAP and Sic1 content was evaluated by immunoblot. Where indicated, arsenite was added to the reaction prior to substrate addition without (lanes 7–10) or with (lanes 11–14) recombinant AIRAP (as indicated). Lanes 1 and 2 (input) are recombinant Sic1 and in vitro polyubiquitinated Sic1 without proteasomes.