1. Regulation of SUMOylation by Reversible Oxidation of SUMO Conjugating Enzymes 
Guillaume Bossis, Frauke Melchior 
Molecular Cell 
Volume 21, Issue 3, Pages (February 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 H2O2 Causes Reversible Loss of SUMO Conjugation
(A) HeLa cells were treated with increasing H2O2 concentrations for 1 hr, lysed in Laemmli buffer, and immunoblotted with α SUMO1, α SUMO2, or α ubiquitin.
(B) Time course of H2O2 treatment. HeLa cells were incubated with 1 mM H2O2 for different times, treated as in (A), run on a 6% SDS-polyacrylamide gel, and immunoblotted with goat SUMO-1 (upper panel) to detect SUMO-1 conjugates; or, samples were run on a 15% SDS-polyacrylamide gel and immunoblotted with mouse monoclonal GMP-1 antibody to detect the free SUMO-1.
(C) Effect of H2O2 on specific SUMO targets. c-Fos and c-Jun expression was induced in HeLa cells by addition of 20% serum for 1 hr. For c-Fos, c-Jun, and RanGAP, 1 mM H2O2 was then added to the medium for the indicated time, and cells were lysed in Laemmli buffer and immunoblotted with specific antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Dose-Dependent Effects of H2O2 on SUMO Conjugation and Deconjugation Machineries
(A) SUMO FRET assay. When YFP-SUMO and CFP-RanGAPtail are not conjugated, excitation of CFP at 430 nm results in strong emission at 485 nm. Upon addition of the conjugating enzymes (Aos1/Uba2 and Ubc9) and ATP, YFP-SUMO is covalently conjugated to CFP-RanGAPtail. This allows FRET to take place. As a consequence, emission at 485 nm is reduced and emission at 527 nm increases.
(B) Inhibition of SUMO conjugation machinery by H2O2. Ubc9 (45 nM) and Aos1/Uba2 (15 nM) were incubated together without (▴) or with 0.1 mM (□) or 1 mM (■) H2O2 for 30 min prior to the addition of YFP-SUMO-1 (250 nM) and CFP-RanGAPtail (250 nM). Addition of ATP (1 mM) started the reaction, and FRET signal was acquired every minute for 30 min.
(C) Inactivation by H2O2 requires coincubation of Aos1/Uba2 and Ubc9. Ubc9 (45 nM) and Aos1/Uba2 (15 nM) were incubated together without (▴) or with 1 mM (■) H2O2 for 30 min, or were treated separately (o) with 1 mM H2O2 for 30 min and mixed just before addition of YFP-SUMO-1, CFP-RanGAPtail, and ATP.
(D) Recovery of conjugating activity by reductants. Ubc9 (45 nM) and Aos1/Uba2 (15 nM) were incubated together with 1 mM H2O2 for 30 min prior to the addition of YFP-SUMO-1, CFP-RanGAP, and either 4 mM DTT (o), 4 mM GSH (▴), or the same volume of water (■). Addition of ATP (1 mM) started the reaction.
(E) The isopeptidase SENP-1 is only moderately susceptible to H2O nM preformed YFP-SUMO-1/CFP-RanGAPtail (YFP-SUMO-1 and CFP-RanGAPtail were incubated for 30 min with Aos1/Uba2, Ubc9, and ATP as in [B], 1 U Apyrase was then added to remove ATP for 10 min) was incubated with SENP-1 (15 nM, catalytic domain only) that was preincubated for 30 min without (▴) or with 1 mM (■) or 10 mM (•) H2O2.
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4. Figure 3
H2O2 Inhibits SUMO Thioester Formation and Leads to a DTT-Sensitive Crosslink between Uba2 and Ubc9 In Vitro
(A) SUMO thioester formation is abolished by H2O2 in vitro. Recombinant E1 (Aos1/Uba2, 100 nM) and Ubc9 (1 μM) were incubated together, with or without H2O2 (1 mM), for 30 min prior to the addition of SUMO-1 (5 μM) and ATP (1 mM). The reaction was stopped by the addition of thioester sample buffer with or without DTT and was analyzed by immunoblotting.
(B) Disulfide bridge formation between Ubc9 and Uba2. Recombinant E1 (Aos1/Uba2, 100 nM) and Ubc9 (1 μM) were incubated alone or together for 30 min with 1 mM H2O2 where indicated. Reactions were stopped as in (A). The star represents a small fraction of DTT-sensitive Ubc9 dimer.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 H2O2 Induces Oxidative Coupling of Uba2 and Ubc9 In Vivo
(A) Effect of H2O2 on SUMO conjugating enzymes. HeLa cells were incubated with 1 mM H2O2 for the indicated times, lysed in Laemmli buffer without (upper panels) or with (lower panels) DTT, and loaded onto 5%–20% SDS-polyacrylamide gels and immunoblotted with Ubc9, Uba2, and Aos1 antibodies.
(B) In vivo inhibition of thioester formation. HeLa cells were incubated, where indicated, with 1 mM H2O2 for 2 min and lysed in Laemmli buffer without or with DTT and immunoblotted with Ubc9 antibodies.
(C) Uba2-Ubc9 crosslinks at different H2O2 concentrations. HeLa cells were incubated for 60 min with the indicated H2O2 concentrations, lysed in Laemmli buffer without DTT, loaded onto a 6% SDS-polyacrylamide gel, and immunoblotted with Uba2 or Ubc9 antibodies. The faint high molecular weight band observed in anti Uba2, but not in anti Ubc9, blots in the absence of H2O2 represents a Uba2-SUMO thioester (Uba2-SUMO thioester comigrates with Uba2-Ubc9-crosslinked species). The star indicates a nonspecific background band.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Regulation of Sumoylation by Endogenous H2O2 Production
(A) Uba2-Ubc9 crosslink in activated macrophages. RAW cells were stimulated with PMA (500 nM) and opsonized zymosan (0.5 mg/ml) or with 1 mM H2O2, lysed in nonreducing Laemmli buffer at different time points after induction, loaded onto a 6% SDS-polyacrylamide gel, and immunoblotted with Ubc9 or Uba2 antibodies.
(B) Endogenous H2O2 production by NADPH oxidase is responsible for Uba2-Ubc9 crosslink. RAW cells were stimulated for 1 hr with opsonized zymosan (0.5 mg/ml), PMA (500 nM), or both; were lysed as in (A), and immunoblotted with Ubc9 (upper panel) or α-tubulin (lower panel) antibodies. Inhibitors (20 mM NAC, 20 μM DPI, 1 mM NMMA) were added 15 min prior to the induction. 1 mM H2O2 was added for 15 min where indicated.
(C) GSH is responsible for Uba2-Ubc9 crosslink reduction. RAW cells were incubated with BSO (300 μM) for 16 hr to deplete the GSH pool prior to induction and analysis as in (B).
(D) Accumulation of free SUMO1 in activated macrophages. RAW cells were stimulated with PMA (500 nM) and opsonized zymosan (0.5 mg/ml) for the indicated times, lysed in Laemmli buffer, loaded onto a 15% SDS-polyacrylamide gel, and immunoblotted with monoclonal anti-SUMO (α GMP-1) antibody (lower panel) or loaded onto a 6% SDS-polyacrylamide gel and immunoblotted with goat polyclonal SUMO-1 antibodies.
(E) Cytoplasmic localization of the oxidized Uba2-Ubc9 species. RAW cells were stimulated for 1 hr with PMA (500 nM) and zymosan (0.5 mg/ml) or for 15 min with 1 mM H2O2 and fractionated to separate cytoplasmic (S100) and nuclear proteins. Cell equivalents were loaded onto 5%–20% SDS-polyacrylamide gels and blotted with Ubc9 (upper panel) and Uba2 (lower panel) antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
H2O2 Induces the Formation of a Disulfide Bond between Uba2 and Ubc9 Catalytic Cysteines
(A) In vitro analysis of Uba2 and Ubc9 cysteine to serine mutants. Recombinant E1 (Aos1/Uba2, 100 nM) and Ubc9 (1 μM) Cys/Ser mutants, as indicated, were incubated for 30 min with or without H2O2 (1 mM) prior to analysis with 6% SDS-PAGE in the absence of DTT and immunoblotting with Uba2 and Ubc9 antibodies.
(B) In vivo disulfide between Uba2 and Ubc9 catalytic cysteines. HeLa cells transiently expressing HA-tagged wild-type or C93S Ubc9 (left panel) or Flag-tagged wild-type or C173S Uba2 (right panel) were incubated for 15 min with 1 mM H2O2, lysed in Laemmli buffer, and immunoblotted with the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Regulation of SUMOylation by Intracellular Redox Status
In normal redox conditions (left panel), there is an equilibrium between SUMO conjugation and deconjugation. Conjugation is achieved through successive thioester bond formation between E1 (Aos1/Uba2) and E2 (Ubc9) catalytic cysteines and SUMO and isopeptide linkage on target lysines. Deconjugation is due to specific isopeptidases, such as SENP family members. When the cellular environment is shifted toward a more oxidizing one (right panel), either globally or locally, SUMO conjugation is reduced due to disulfide bridge formation between Uba2 and Ubc9 catalytic cysteines. Because the SUMO-isopeptidases are not affected unless exceedingly high concentrations of ROS are present, a shift in the SUMO conjugation-deconjugation balance toward desumoylation of modified proteins is induced.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions