Volume 47, Issue 1, Pages 76-86 (July 2012) A Mutually Inhibitory Feedback Loop between the 20S Proteasome and Its Regulator, NQO1  Oren Moscovitz, Peter Tsvetkov, Nimrod Hazan, Izhak Michaelevski, Hodaya Keisar, Gili Ben-Nissan, Yosef Shaul, Michal Sharon  Molecular Cell  Volume 47, Issue 1, Pages 76-86 (July 2012) DOI: 10.1016/j.molcel.2012.05.049 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 MS Analysis Reveals that NQO1 Is a Homodimer that Exists Either in Apo Form or Bound to One or Two FAD Molecules (A and B) Mass spectra of NQO1 prior to (A) and following (B) the addition of 50 μM FAD for 5 min at room temperature. The measured mass of NQO1 indicates that it is a dimer in which the relative abundance of the apo and holo forms depends on FAD levels. Colored dots designate the different numbers of bound FAD molcules. See also Figure S1. Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 In the Absence of FAD Binding, NQO1 Is Partially Unfolded (A–D) MS spectra of NQO1∗2 (A), NQO1∗2 after incubation with 200 μM FAD for 1 hr at 4°C (C), and NQO1 after extraction of the FAD cofactor (apo-NQO1) (D). Both apo-NQO1 and NQO1∗2 display a wide distribution of charge states (14+–29+) compared to wtNQO1 (Figure 1) or to NQO1∗2 incubated with FAD (C). This heterogeneous, highly charged population indicates the existence of partially unfolded conformers. Spectra were acquired in 500 mM ammonium acetate, pH 7.4. Colored dots indicate the charge state series corresponding to dimeric NQO1 bound to different numbers of FAD molecules. Red stars represent ubiquitin, which was used as an internal standard. Black asterisks correspond to a nonrelated bacterial protein which is copurified with NQO1. (B) Calculated CCS values obtained from IM-MS experiments of wtNQO1 and NQO1∗2. The high charge states of NQO1∗2 display a significantly larger CCS value compared to the lower charge states (14+–17+) or to the CCS values calculated for wtNQO1. Error bars represent SD for four different wave heights. (E) Thermal stability assays of apo-NQO1, wtNQO1, and NQO1∗2 as well as NQO1 and NQO1∗2 saturated with 250 μM FAD. Samples were incubated for 10 min at a range of temperatures, followed by cooling to room temperature and measurement of activity levels. The shifts in T50 between the apo and holo forms reflect the structural stabilization induced by FAD binding. (F) Activity assays of apo-NQO1, wtNQO1, NQO1∗2, as well as NQO1 and NQO1∗2 saturated with 250 μM FAD. The reaction monitored the reduction of 42 μM of 2,6-dichloroindophenol. The absorbance at 600 nm was measured after 15 min at room temperature. The curves indicate that saturating the FAD binding sites of both WT and mutant forms of NQO1 significantly increases the reductase activity of the protein. Error bars in (E) and (F) represent the SD of four repeats. See also Figure S2. Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 NQO1 Binds to the 20S Proteasome in a NADH-Independent Manner (A–D) Prior to MS analysis, the 20S proteasome was incubated with NQO1 (overnight at 4°C, at a ratio of 10:1, NQO1:20S), with (B) or without (C) 1 mM NADH. As a control, both free 20S proteasome (A) and NQO1 (D) were examined. For each sample, the most intense charge state obtained in the MS spectrum was subjected to MS/MS analysis (inset shows MS spectrum of the free 20S proteasome; the 64+ charge state highlighted in red was subjected to MS/MS analysis). Blue and cyan dots correspond to two different α-subunits of the 20S; green dots represent monomers of NQO1. See also Figure S3. Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Apo-NQO1 Is a Substrate of the 20S Proteasome (A) In vitro 20S proteasome degradation assays of NQO1∗2, apo-NQO1, wtNQO1, and NQO1 saturated with 250 μM FAD and apo-NQO1 in the presence of 2 mM Velcade. We incubated 4.5 μg of each NQO1 form with the 20S proteasome at 37°C in a 1:1 molar ratio. At different time points, the reaction was stopped by the addition of Laemmli sample buffer. The mixture was then heated at 95°C for 5 min and analyzed by immunoblotting with anti-NQO1 antibody. (B) NQO1∗2 was incubated at a ratio of 1:1:1.5, 20S proteasome:NQO1:ubiquitin and subjected to MS analysis. The three panels represent three different time points. Green dots correspond to NQO1∗2, and red stars represent ubiquitin, which was used as an internal control. (C) Apo-NQO1, wtNQO1, and NQO1∗2 were incubated as follows: with or without the 20S proteasome, in the presence or absence of 500 μM FAD, and at a ratio of 7:1 (NQO1:20S proteasome) for 3 hr at 30°C. Generated peptides were collected by passing the samples through a C18 ZipTip. The peptides were identified by proteomic analysis. See also Figure S4. Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Apo-NQO1 Is Susceptible to Degradation by the 20S Proteasome We incubated 3.5 μg of wtNQO1 with the 20S proteasome at 30°C at a ratio of 4:1 (20S proteasome:NQO1) and subjected it to MS analysis at several time points. The change in the relative distribution of the NQO1 FAD-bound forms clearly indicates a reduction in apo-NQO1 levels. As a control, NQO1 was incubated at 30°C in the absence of the proteasome (bottom panel). For visualization purposes, we added a red box to highlight the different NQO1 forms within a single charge state (16+). Colored dots correspond to dimeric NQO1, with different numbers of bound FAD molecules. Black asterisks correspond to a nonrelated bacterial protein that is copurified with NQO1. Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 NQO1 Is Susceptible to 20S Degradation in the Cellular Environment, but Can Be Stabilized by FAD (A) In vitro translated [35S] methionine-labeled wtNQO1 and NQO1∗2 were incubated at 37°C in the presence of purified 20S proteasomes at the indicated time points. [35S]-labeled proteins were detected by autoradiography. (B) 20S proteasomal degradation of wtNQO1 and NQO1∗2 was analyzed following their incubation at 37°C for 60 min in the presence of increasing concentrations of FAD (as indicated). (C and D) HEK293 cells were transfected with either wtNQO1 (C) or NQO1∗2 (D) and their cellular levels were determined as a function of riboflavin concentration. (E–G) The levels of NQO1, NQO1∗2, p53, and Hsc70 (as loading control) were examined, following treatment with indicated concentrations of riboflavin for 12 hr. Control HeLa cells (G) or HeLa cells overexpressing either wtNQO1 (E) or NQO1∗2 (F) were treated with increasing concentrations of riboflavin for 12 hr, after which the levels of NQO1 and Hsc70 were analyzed. (H) MDA-MB-231 cells were treated for 16 hr with increasing concentrations of riboflavin. In all panels protein levels were detected by immunoblotting with anti-NQO1, anti-p53, anti-Hsc70, and anti-Actin antibodies. Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 A Mutually Inhibitory Feedback Loop Exists between NQO1 and the 20S Proteasome NQO1 can prevent the proteolytic activity of the 20S proteasome, while the proteasome can degrade apo-NQO1, which adopts a partially unfolded conformation. The levels of apo-NQO1 in the cell depend on the free FAD levels, thus linking NQO1 activity to the metabolic status. Molecular Cell 2012 47, 76-86DOI: (10.1016/j.molcel.2012.05.049) Copyright © 2012 Elsevier Inc. Terms and Conditions