1. Redox-Dependent Control of FOXO/DAF-16 by Transportin-1
Marrit Putker, Tobias Madl, Harmjan R. Vos, Hesther de Ruiter, Marieke Visscher, Maaike C.W. van den Berg, Mohammed Kaplan, Hendrik C. Korswagen, Rolf Boelens, Michiel Vermeulen, Boudewijn M.T. Burgering, Tobias B. Dansen 
Molecular Cell 
Volume 49, Issue 4, Pages (February 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 49, 730-742DOI: (10.1016/j.molcel.2012.12.014)
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3. Figure 1
A Mass-Spectrometry-Based Screen Identifies Several Disulfide-Dependent Binding Partners of FOXO4
(A) The setup of the screen. Flag-FOXO4 cysteine mutants overexpressing cells were incubated with 200 μM H2O2 30 min before lysis. Flag-FOXO4 pull-downs were stringently washed and their binding partners were identified in a quantitative (1) and qualitative (2) tandem-mass-spectrometry (MS/MS)-based manner.
(B) A volcano plot of the quantitative MS/MS data showing statistical significance versus fold change. The logarithmic ratio of protein intensities of pull-downs of FOXO4Cys239 over FOXO4ΔCys were plotted against negative logarithmic p values of the t test performed from quadruplicates. Proteins with a Log2 (single Cys/ΔCys) ratio higher than 2 (4-fold) and a p value lower than 0.05 were considered cysteine-dependent binding partners of FOXO4. The horizontal red line corresponds to a p value cutoff of The green vertical lines correspond to a 4-fold change.
See also Figure S1.
(C) Nonreducing SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis of immunoprecipitated Flag-FOXO4 cysteine mutants. HEK293T cells were incubated with 200 μM H2O2 30 min before lysis. The upper panel shows western blots of ∼10% of each sample stained for Flag that identify Flag-FOXO4 and potential disulfide-dependent Flag-FOXO4 containing complexes that migrate at higher molecular weight (#). The middle panel shows Simply Blue-stained gel (∼90% of each sample) that was used for the isolation of peptides for MS/MS analysis. The boxed bands are used for label-free quantification (LFQ) analysis in Figure 2A. The lower panel shows whole cell lysates of the samples used in the qualitative screen and shows that the expression of different mutants was equal.
See also Figure S2 and Table 1.
(D) Western blot analysis of Flag-FOXO4 immunoprecipitates confirms the preference of certain FOXO4 cysteines for some of the hits found in the MS/MS screen. USP7 and TNPO1 were identified as disulfide-dependent binders in the secondary MS screen, whereas insulin degrading enzyme (IDE) was not detected in the secondary screen, but the interaction is clearly both cysteine-dependent and induced by H2O2 treatment. Note that the TNPO1 antibody stains both a specific and a nonspecific band; the lower of the two bands (∗) is TNPO1.
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4. Figure 2 TNPO1 Is a Disulfide-Dependent Interaction Partner of FOXO
(A) LFQ of TNPO1 pulled down in the boxed bands indicated in Figure 1D confirms that TNPO1 binds preferentially to cysteine 239.
(B) Endogenous FOXO4-TNPO1 complexes can be identified in HEK293T cells upon applying H2O2. Nonimmune serum (N.I.) was used as a control.
(C) Endogenous ROS, produced when U2OS cells are deprived of glucose, is sufficient to induce FOXO4-TNPO1 complex formation. Incubation of the cells with the antioxidant N-acetylcysteine (NAC) prevents complex formation. Green fluorescent protein (GFP) expression was used as a negative control. Samples shown are from the same western blot. Dashed lines indicate digital rearrangement of lanes.
(D) Redox-sensitive binding of TNPO1 is conserved between FOXO homologs.
(E) FOXO4-TNPO1 complexes are dependent on cysteine-thiol oxidation, as the ROS-induced binding is lost when YFP-TNPO1 immunoprecipitates are incubated with 10 mM DTT for 15 min prior to washing with high-salt buffer.
(F) Parallel reducing and nonreducing SDS-PAGE with subsequent western blot analysis of Flag-FOXO4 (green) and TNPO1 (red). Upon H2O2 treatment, Flag-FOXO and TNPO1 migrate as one complex at the molecular weight of FOXO4+TNPO1 (∼160kDa) when studied under nonreducing conditions. The covalent interaction is lost when samples are incubated with 10 mM DTT prior to washing, or when the samples are studied under reducing conditions.
See also Figures S3 and S4.
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5. Figure 3
NMR Analysis of the Redox-Dependent Interaction of FOXO4 with TNPO1
(A) Superposition of 2D 1H,15N HSQC spectra of FOXO4 (residues 200–505) free (black) and bound to 0.3 stoichiometric equivalents of unlabeled TNPO1 (green) recorded at different redox conditions. NMR signals of FOXO4 residues that are affected by the binding of TNPO1 are encircled. Selected signals are enlarged and shown in separate boxes. Under reducing conditions (left spectrum), the interacting FOXO4 NMR signals are extensively broadened. Under mild oxidizing conditions (middle spectrum), 0.3 stoichiometric equivalents of FOXO4 are locked in the FOXO4-TNPO1 complex while the 0.7 stoichiometric equivalents remain unbound. This gives rise to the reappearance of NMR signals of unbound FOXO4 that have reduced signal intensity according to the amounts of both free FOXO4 and FOXO4 locked in the high-affinity complex (see also Figure S5). Formation of the high-affinity complex is reversed by the addition of 5 mM DTT (right spectrum).
(B) A diagram summarizing the results of the NMR experiments for the FOXO4-TNPO1 complexes formed under different redox conditions.
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6. Figure 4
TNPO1 Is Required for Rapid FOXO4 Nuclear Localization and Activation upon Increased ROS Levels
(A) Immunofluorescence microscopy images showing typical examples of nuclear and cytoplasmic localization of HA-FOXO4. U2OS cells were transfected with scrambled (Scr.) or TNPO1 RNAi oligos. Cells were pretreated with insulin and subsequently incubated for 45 min with or without 100 μM H2O2 in the presence of Leptomycin B (LMB) to block nuclear export. Scale bar represents 20 μm.
(B) H2O2-induced nuclear localization of FOXO4 requires TNPO1, but nuclear localization of FOXO4 as a result of treatment with the PKB inhibitor VIII does not require TNPO1. Quantification of a typical experiment (n = 3). Slides were blinded, N ≥ 200 cells (scored as in Figure S6A). ∗, significant difference. p values were calculated in a two-tailed Fischer’s exact test with the use of a 2×2 contingency table.
(C) Knockdown of tnpo1 was checked by western blot.
(D) Cysteines in FOXO4 are required for efficient FOXO4 translocation. Quantification of the percentage of cells showing translocation within the duration of time-lapse movies (∼70 min after addition of treatments) of Tet-on GFP-Flag-FOXO4 and GFP-Flag-FOXO4ΔCys cells. Basal nuclear localization is lower than in (B), possibly due to the stable, very low levels of FOXO compared to transient overexpression. ∗, significant difference. p values were calculated in a two-tailed Fischer’s exact test with the use of a 2×2 contingency table.
(E) H2O2-induced nuclear translocation of FOXO4ΔCys occurs later than that of FOXO4. Quantification of the timing of GFP-FOXO4 translocation in the same experiment as shown in (D). 500 μM H2O2 was added at time point 0. The time point at which nuclear accumulation had proceeded until the intensity of FOXO4 in the cytoplasm was indistinguishable from the intensity in the nucleus is plotted per cell. ∗, significant difference in an unpaired t test (n = 4, a typical experiment is shown).
(F) Chromatin fractions of H2O2- or VIII (PKB inhibitor)-treated Tet-on GFP-Flag-FOXO4 and GFP-Flag-FOXO4ΔCys cells (all samples were Dox-treated) show that H2O2-induced chromatin association of FOXO4ΔCys is reduced when compared to FOXO4, whereas it is equal in PKB-inhibited cells. The lower panel shows a quantification of the western blots, corrected over the loading control Histone H3. Controls for the efficiency of separation of samples in cytoplasmic and chromatin fractions are included in Figure S6E.
(G) Delayed nuclear translocation and chromatin association of FOXO4ΔCys correlates with a delayed induction of expression of the FOXO4 target gene sod2. PKB-inhibition-induced gene expression (VIII) by FOXO4ΔCys is equal to that of wild-type (WT) FOXO4. Data are represented as mean ±SD of three independent experiments. ∗, p < 0.05 when WT is compared to ΔCys. #, < 0.05 when treated cells are compared to mock-treated cells and tested in an unpaired t test.
See also Figure S6.
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7. Figure 5
IMB-2 Is Required for Stress-Induced Nuclear Localization of DAF-16
(A) Flag-DAF-16 expressed in HEK293T cells, such as FOXO4, interacts with TNPO1 in a cysteine- and H2O2-sensitive manner.
(B and C) Typical localization of DAF-16::GFP in adult TJ356 worms under (B) normal and (C) stressed conditions; i.e., 2 hr incubation at 33°C. Scale bar represents 200 μm.
(D) Quantification of paraquat-, heat-stress-, and starvation-induced translocation of DAF-16::GFP in adult TJ356 worms grown on control (L4440) or imb-2 RNAi plates shows that IMB-2 is required for stress-induced, but not starvation-induced, translocation of DAF-16::GFP. n ≥ 25. ∗, significant difference.
See also Figure S7B–S7I.
(E) Knockdown of imb-2 was checked by western blot.
(F) IMB-2 coimmunoprecipitates with DAF-16::GFP in a redox-dependent manner upon heat stress as IMB-2 binding is lost upon incubation of DAF-16::GFP immunoprecipitates with DTT before washing.
See also Figure S7.
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8. Figure 6
A Model Integrating the Effects of Insulin, JNK, and TNPO1 Signaling on FOXO4 Localization
FOXO4 localization is regulated via a network of different signaling pathways. The effects of these signaling pathways can be independent of cysteines in FOXO4 (red and black arrows) or dependent on cysteines in FOXO4 (black arrow). Under basal (high growth factor) conditions in our experiments, FOXO4 localizes in the cytoplasm, due to the active nuclear export of FOXO4 by Crm1 that counteracts a low import by an unknown import protein (IMP X); incubation of cells with export inhibitor Crm1 (LMB) causes FOXO to accumulate in the nucleus (Biggs et al., 1999), showing that active import is still ongoing (left). When insulin signaling is inhibited (by starvation or by incubation with PKB inhibitor VIII), FOXO4 accumulates in the nucleus by the action of IMP X (middle), which is no longer counteracted by Crm1 because this requires binding to FOXO4, which depends on PKB-dependent phosphorylation of FOXO4. Under conditions of high ROS levels (right), FOXO4 forms a cysteine-dependent complex with TNPO1 and active import takes place. Furthermore, nuclear export is inhibited, possibly due to the action of c-Jun N-terminal kinase (JNK), leading to the rapid nuclear accumulation of FOXO4 in a cysteine-dependent manner. At the same time, (cysteine-independent) basal nuclear import is still ongoing and, combined with inhibited Crm1, it eventually causes FOXO4ΔCys to accumulate in the nucleus, albeit at much slower rates than WT FOXO4.
Molecular Cell  , DOI: ( /j.molcel )
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