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Quantifying the Global Cellular Thiol–Disulfide Status

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Quantifying the Global Cellular Thiol–Disulfide Status Quantifying the global cellular thiol–disulfide status Rosa E. Hansen, Doris Roth, and Jakob R. Winther1 Department of Biology, University of Copenhagen, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark Communicated by Jonathan Beckwith, Harvard Medical School, Boston, MA, November 29, 2008 (received for review July 25, 2008) It is widely accepted that the redox status of protein thiols is of central specific proteins (6–10). Although these studies have supplied importance to protein structure and folding and that glutathione is an pieces to the puzzle, the global picture of the distribution between important low-molecular-mass redox regulator. However, the total reduced and oxidized thiols in proteins have not been determined. cellular pools of thiols and disulfides and their relative abundance Furthermore, the relative abundance of GSH and PSH, and the have never been determined. In this study, we have assembled a total amount of PSSG, under normal conditions is unknown. To fill global picture of the cellular thiol–disulfide status in cultured mam- these gaps, we have developed a new methodology for the quan- malian cells. We have quantified the absolute levels of protein thiols, titative determination of the absolute thiol and disulfide levels in all protein disulfides, and glutathionylated protein (PSSG) in all cellular proteins, including membrane proteins, in cultured mammalian protein, including membrane proteins. These data were combined cells. These data are combined with quantifications of GSH and with quantification of reduced and oxidized glutathione in the same GSSG in the same cells. Furthermore, by exposing cells to the cells. Of the total protein cysteines, 6% and 9.6% are engaged in thiol-specific oxidant diamide and quantifying the redistribution of disulfide bond formation in HEK and HeLa cells, respectively. Further- the thiol and disulfide pools we show that PSH constitutes a pool more, the steady-state level of PSSG is <0.1% of the total protein of active reducing equivalents that is larger than that of GSH. cysteines in both cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels Results increase to >15% of all protein cysteine. Glutathione is typically Experimental Approach. The experimental strategy for quantifica- characterized as the ‘‘cellular redox buffer’’; nevertheless, our data tion of cellular redox species is illustrated in Fig. 1A. A crucial point, show that protein thiols represent a larger active redox pool than when working with redox biology, is to avoid oxidation and thiol– glutathione. Accordingly, protein thiols are likely to be directly in- disulfide exchange during sample preparation. The combined acid- volved in the cellular defense against oxidative stress. ifying and denaturing effects of trichloroacetic acid (TCA) makes it an ideal choice for quenching redox reactions in cell extracts. cysteine ͉ glutathione ͉ protein Furthermore, addition of TCA to 10% results in protein precipi- tation, whereas low-molecular-mass metabolites stay soluble. ecause of the thiol group (SH), cysteine is the most chemically To selectively quantify the different protein sulfhydryl species reactive natural amino acid found in cells. SH is mainly found TCA pellets are divided into fractions and solubilized by sonicating B in buffers appropriate for thiol and disulfide quantifications. Im- in proteins (PSH) and in low-molecular-mass metabolites such as portantly, this procedure renders even thiols that are buried in the the highly abundant glutathione (GSH). Two SH groups can be native protein structures accessible to the thiol detection agent. oxidized to form a disulfide bond, and, in the cell, disulfides are Thiol detection reactions are generally dependent on the de- mainly found in proteins (PSSP), in glutathione disulfide (GSSG), protonated thiolate anion, and because pK values of SH groups are or as mixed disulfides between protein and glutathione (PSSG). In a typically Ϸ8–9, a pH of at least 7 is usually required for these addition, SH groups can be reversibly oxidized by reactive oxygen reactions to occur. At this pH, however, unwanted spontaneous species to nitrosothiols or sulfenic acids. Higher oxidation states, redox reactions, such as oxidation, take place on the microsecond such as sulfinic and sulfonic acids, are generally considered to be time scale (11). To circumvent this potential problem we have irreversible. quantified protein sulfhydryl equivalents by use of the thiol specific Disulfides are important for protein structure and folding, and reagent 4-dithiodipyridine (4-DPS), which reacts with thiols to form formation of disulfide bonds can regulate protein function (1). stoichiometric amounts of the chromogenic compound 4-thiopyri- Furthermore, glutathionylation, here used to denote the formation done (4-TP), absorbing at 324 nm (Fig. 1B). In this context, the of PSSG, has been implicated as a mechanism for protection against primary virtue of 4-DPS is its reactivity at low pH where thiol– irreversible protein oxidation during oxidative stress (2). disulfide reactions do not normally occur (12). The cytosolic glutathione pool is highly reducing (3) and conse- Although detection of thiols is fairly straightforward, accurate quently, disulfide bond formation is infrequent and typically only quantification of disulfides is more complicated as it requires takes place transiently during catalysis of redox processes. In alkylation of free thiols followed by reduction of disulfides and their contrast, disulfide bond formation is favored in other compart- subsequent quantification. However, reagents for disulfide reduc- ments of the cell, such as the endoplasmic reticulum (ER) and the tion, thiol alkylation, and thiol quantification are frequently found intermembrane space of mitochondria (4). The overall distribution to cross-react. Disulfide levels can, for example, be underestimated of thiols and disulfides in different cellular pools defines the global if left-over alkylating agent cross-reacts with newly reduced thiols. thiol–disulfide redox status of the cell. Maintenance of thiol– In contrast, overestimation of disulfide amounts can occur, if the disulfide redox homeostasis in different subcellular compartments reducing agent cross-reacts with the quantification reagent. is important, and failure to do so can be detrimental to the To selectively quantify disulfides involving proteins (PSox), we structure, stability and activity of various proteins, including en- alkylated with N-ethylmaleimide (NEM) followed by reduction zymes, chaperones, and transcription factors. Over recent years several methods have been developed for studying the protein thiol status of the cell. These methods typically Author contributions: R.E.H. and J.R.W. designed research; R.E.H. and D.R. performed involve differential labeling of protein thiols and disulfides, fol- research; R.E.H. and J.R.W. analyzed data; and R.E.H. and J.R.W. wrote the paper. lowed by chromatographic or electrophoretic separation of cellular The authors declare no conflict of interest. proteins. Finally, cysteine containing proteins are identified by mass 1To whom correspondence should be addressed. E-mail: [email protected]. spectrometry and in some cases information of the thiol–disulfide This article contains supporting information online at www.pnas.org/cgi/content/full/ status of peptides are also determined (5, 6). Some studies have thus 0812149106/DCSupplemental. quantified changes in the ratio of reduced to oxidized thiols in © 2009 by The National Academy of Sciences of the USA 422–427 ͉ PNAS ͉ January 13, 2009 ͉ vol. 106 ͉ no. 2 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812149106 Downloaded by guest on September 25, 2021 A Cells A B 95 % Confluent plate 120 120 Trypsinization 100 100 80 80 Acidification/Protein precipitation 10% TCA 60 60 40 40 Fraction of PS (%) Fraction of PS (%) Fraction Supernatant: Pellet: 20 20 LMW quantification Protein quantification 0 0 Total Total Total Total PScal PSexp PScal PSexp S1 S2 P1 P2 P3 P4 Total GS GSSG PSH PSox Total PS PSSG Fig. 2. Validation of the method for quantification of cellular protein thiols (SQ/PQ) (SQ/PQ) (SQ/PQ) (SQ/PQ) (SQ/PQ (SQ/PQ) and disulfides. Percentage of measured SH/aa relative to the calculated value of total SH/aa (Total PScal) is shown. (A)%PSox (gray bar) is stacked with % PSH C (white bar). This bar shows Total PScal. The black bar shows the value of the B experimentally determined total PS (Total PSexp). (B) P2 and P3 samples (de- fined in Fig. 1A) were spiked with equal amounts of purified lysozyme, which contains 4 disulfide bonds. Exogenously added lysozyme PS equivalents (PSlys) in P2 and P3 were recovered by (100 Ϯ 5)% and (106 Ϯ 3)%, respectively. The percentage of PSH (white bar) is stacked with the percentage of (PSox ϩ PSlys) SH SX SH SH shown in the gray bar. The black bar shows percentage of (Total PSexp ϩ PSlys). SSG SSG SSG SSG S S S S Finally, PSSG was quantified by reduction of disulfide bonds with S S S S tris(hydroxypropyl)phosphine (THP) and fluorescent labeling of sulfhydryl compounds with 7-fluorobenzo-2-oxa-1,3-diazole-4- sulfonate (SBD-F; (14)) by incubating at pH 8.5 at 60 °C for1h(Fig. 1C). By applying HPLC with fluorescence detection the SBD-GS 4-TP thiol SX SH S-SBD quantification derivative can be selectively determined. The method exploits that SH GSH SH GSH S-SBD GS-SBD phosphines do not cross-react with SBD-F, and reduction and derivatization can take place in the same test tube (15). This means that SH SH S-SBD SH SH S-SBD the rather harsh reaction conditions do not cause artificial thiol oxida- tion, because reducing agent is present during the derivatization. In addition to sulfhydryl quantification, the total protein content in the pellet fractions were determined (Fig. 1A) and used as a GS-SBD common denominator to compare the individual samples. This is a quantification crucial step as it eliminates any bias from a possible uneven division Fig.
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