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Sulfenome Mining in Arabidopsis Thaliana

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Sulfenome Mining in Arabidopsis Thaliana Sulfenome mining in Arabidopsis thaliana Cezary Waszczaka,b,c,d,e,1, Salma Aktera,b,c,d,e,f,1, Dominique Eeckhouta,b, Geert Persiaua,b, Khadija Wahnic,d,e, Nandita Bodraa,b,c,d,e, Inge Van Mollec,d,e, Barbara De Smeta,b,c,d,e, Didier Vertommeng, Kris Gevaerth,i, Geert De Jaegera,b, Marc Van Montagua,b,2, Joris Messensc,d,e,2, and Frank Van Breusegema,b,2 aDepartment of Plant Systems Biology, VIB, 9052 Ghent, Belgium; bDepartment of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; cStructural Biology Research Center, VIB, 1050 Brussels, Belgium; dBrussels Center for Redox Biology, 1050 Brussels, Belgium; eStructural Biology Brussels Laboratory, Vrije Universiteit Brussel, 1050 Brussels, Belgium; fDepartment of Fisheries, Faculty of Biological Sciences, University of Dhaka, 1000 Dhaka, Bangladesh; gde Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium; hDepartment of Medical Protein Research, VIB, 9000 Ghent, Belgium; and iDepartment of Biochemistry, Ghent University, 9000 Gent, Belgium Contributed by Marc Van Montagu, June 29, 2014 (sent for review April 9, 2014) Reactive oxygen species (ROS) have been shown to be potent develop a YAP1-based probe for trapping plant sulfenylated signaling molecules. Today, oxidation of cysteine residues is proteins in vivo (12). a well-recognized posttranslational protein modification, but the To categorize the sulfenome, which is the set of proteins signaling processes steered by such oxidations are poorly un- with at least one sulfenic acid, and its dynamics at the proteome derstood. To gain insight into the cysteine thiol-dependent ROS level in Arabidopsis thaliana cells upon oxidative stress, we imple- signaling in Arabidopsis thaliana, we identified the hydrogen per- mented the YAP1-based sulfenic acid trapping method coupled oxide (H2O2)-dependent sulfenome: that is, proteins with at least to a tandem affinity purification (TAP) tag (13). We identified one cysteine thiol oxidized to a sulfenic acid. By means of a genetic 97 sulfenylated proteins during the early and late oxidative construct consisting of a fusion between the C-terminal domain of stress responses, of which 67 had previously not been recognized Saccharomyces cerevisiae – the yeast ( ) AP-1 like (YAP1) transcrip- to undergo oxidative PTMs. Validation of sulfenylation on tion factor and a tandem affinity purification tag, we detected DEHYDROASCORBATE REDUCTASE2 (DHAR2) demon- ∼ Arabidopsis 100 sulfenylated proteins in cell suspensions ex- strates the importance of a glutathione (GSH)-dependent redox posed to H2O2 stress. The in vivo YAP1-based trapping of sulfeny- switch on its sulfenylated nucleophilic cysteine that reversibly lated proteins was validated by a targeted in vitro analysis of regulates the DHAR activity. DEHYDROASCORBATE REDUCTASE2 (DHAR2). In DHAR2, the ac- tive site nucleophilic cysteine is regulated through a sulfenic Results and Discussion acid-dependent switch, leading to S-glutathionylation, a protein H O Triggers the Formation of YAP1C Heterocomplexes in a Time- and modification that protects the protein against oxidative damage. 2 2 Dose-Dependent Manner. To apply the YAP1-TAP approach, we synthesized a YAP1-cCRD construct with adapted codons for oxidative stress | redox regulation | cysteine oxidation proficient expression in plants and mutated Cys620 and Cys629 to alanine and threonine (Fig. S1B and Table S1), retaining only umerous posttranslational modifications (PTMs) have been the redox-active cysteine Cys598. Then, we fused this construct PLANT BIOLOGY Ndiscovered within proteomes, creating a complex landscape at its N terminus to a GS tag that combines two IgG-binding of protein diversity and function (1). One of the recognized re- domains of protein G with a streptavidin-binding peptide (SBP), versible redox-based PTMs is the oxidation of a cysteine thiol separated by a tobacco etch virus (TEV) protease cleavage site group to a sulfenic acid (Cys-SOH) (2) that acts as regulatory (13). The Cys598 of YAP1C-GS is essential for the formation switch in several oxidative stress signal transduction pathways of mixed disulfides with sulfenylated proteins (12). In addition, (3). Sulfenic acids, unless they are stabilized into the protein we constructed a similar control version, YAP1A-GS, in which environment, can react rapidly with other protein thiols or with low-molecular weight thiols to form intramolecular and in- Significance termolecular disulfides. These mechanisms protect the sulfenic acids against overoxidation to sulfinic (SO2H) or sulfonic (SO3H) acid and allow sulfur oxygen signaling (2). When oxygen gets incompletely reduced, reactive oxygen In plants, the best-known redox regulation mechanisms are the species (ROS) are generated. These ROS molecules can harm the building blocks of the cell but are also important signaling light-dependent thiol-disulfide exchange switches in chloroplast molecules. Until now, the ROS language of the cell has not proteins (4). Examples of other redox-regulated proteins are the been understood and a clear view is needed on how the cell transcription coactivator NONEXPRESSER OF PR GENES 1 + differentiates metabolic ROS noise from ROS that allows sig- (5), the vacuolar H -ATPase (6), and several transcription factors naling, regulation, and protection. To address this question, we (TFs), such as the AP2-type RAP2.4a (7), the G-group of basic focused on Arabidopsis thaliana and identified the proteins leucine zipper TFs (8), and the TEOSINTEBRANCHED1/ that react with hydrogen peroxide on the thiol of the amino CYLOIDEA/PROLIFERATING CELL FACTOR class I TFs acid cysteine, which after reaction forms a sulfenic acid. The (9). The redox relay mechanisms that bridge the signal perception characterization of the plant sulfenome improves the un- to the final oxidative stress response are largely unknown. derstanding of important ROS signaling pathways. Some thiol peroxidases have an H2O2-dependent signaling function and can act as receptor and transducer (10). In yeast Author contributions: C.W., S.A., G.P., I.V.M., M.V.M., J.M., and F.V.B. designed research; C.W., S.A., D.E., G.P., K.W., N.B., I.V.M., and B.D.S. performed research; D.V., K.G., and G.D.J. (Saccharomyces cerevisiae), the H2O2 sensor oxidant receptor contributed new reagents/analytic tools; C.W., S.A., B.D.S., J.M., and F.V.B. analyzed data; peroxidase1 (ORP1/glutathione peroxidase 3) controls, together and C.W., S.A., B.D.S., J.M., and F.V.B. wrote the paper. – with the transcription factor YAP1 (yeast AP-1 like), a redox The authors declare no conflict of interest. regulon via a sulfenic acid thiol-disulfide relay mechanism (11). 1C.W. and S.A. contributed equally to this work. Upon reaction with H O , the peroxidatic cysteine of ORP1 is 2 2 2To whom correspondence may be addressed. Email: [email protected], oxidized to a sulfenic acid that reacts with the YAP1 C-terminal [email protected], or [email protected]. A cysteine-rich domain (cCRD) and forms a disulfide (Fig. S1 ). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. This specific mixed disulfide formation has prompted us to 1073/pnas.1411607111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1411607111 PNAS | August 5, 2014 | vol. 111 | no. 31 | 11545–11550 Downloaded by guest on September 25, 2021 all cysteines were mutated (Fig. S1B and Table S1). The cau- tool to study time- and dose-dependent H2O2 stress responses in liflower mosaic virus 35S promoter-driven constructs were plant cells. transformed in Arabidopsis cell suspensions. Western blot analysis with a specific antibody complex to detect the G Unique Sulfenylated Proteins Are Selectively Trapped with YAP1C-GS. moiety of the tag [peroxidase-antiperoxidase (PAP) antibody To evaluate the early signaling events in the presence of 1 mM complex] revealed that the yield of the two fusion proteins H2O2, we decided to focus on cysteine oxidation 10 min after YAP1C-GS and YAP1A-GS is comparable and that they mi- stress. Protein extracts of cells containing YAP1C-GS and grate as a single band at 35 kDa (Fig. S1C). YAP1A-GS were purified by TAP (Fig. 2). Briefly, first, YAP1-GS Previously, a 20-mM H2O2 treatment of Arabidopsis cells had complexes were captured on IgG-Sepharose. Second, after a TEV been found to provoke oxidative stress signaling (14). We treated protease cleavage step, the YAP1 fused to the SBP tag together the transformed Arabidopsis cell suspension cultures with 0, 0.1, with several mixed disulfide complexes and all possible interacting 1, 5, 10, and 20 mM H2O2 at the mid-log phase for 1 h. To block proteins were eluted. In a subsequent SBP purification step, the all free thiols, we extracted the soluble protein in the presence of mixed disulfide YAP1 complexes were enriched. The sulfenylated iodoacetamide and N-ethylmaleimide (NEM) (Fig. S2) and an- proteins were released by selective elution of the disulfide-bonded alyzed the disulfide bond formation on a nonreducing Western proteins with 5 mM DTT followed by a 20-mM desthiobiotin blot with the PAP antibody complex. In untreated cells and in elution. The majority of interactors (41 of 46) eluted with DTT cells treated with 0.1 mM H2O2, YAP1C-GS migrated at 35 kDa, (Dataset S1). To guarantee a complete interactor recovery, we but H2O2 treatments ranging between 1 and 20 mM resulted in decided to use desthiobiotin for all of the TAP purifications. a proportional increase in the number of high-molecular weight Hereafter, we focused on the late-response sulfenome observed YAP1C-GS complexes (Fig. 1A). The disulfide nature of the 1 h after an oxidative stress pulse with 0, 1, and 20 mM H2O2.In interactions has been proven by the disappearance of most of the agreement with the increased mixed-disulfide complex formation high-molecular weight bands on a reducing Western blot (Fig.
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