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Originally published In Press as doi:10.1074/jbc.M200591200 on March 29, 2002

J. Biol. Chem., Vol. 277, Issue 24, 21189-21196, June 14, 2002
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S-Thiolation of HSP27 Regulates Its Multimeric Aggregate Size Independently of Phosphorylation*

Philip EatonDagger, William Fuller, and Michael J. Shattock

From The Centre for Cardiovascular Biology and Medicine, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, United Kingdom

Received for publication, January 18, 2002, and in revised form, March 5, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HSP27 exists as large aggregates that breakdown after phosphorylation. We show rat cardiac HSP27 is S-thiolated during oxidant stress, and this modification, without phosphorylation, disaggregates multimeric HSP27. Biotinylated cysteine acts as a probe for thiolated proteins, which are detected using non-reducing Western blots probed with streptavidin-horseradish peroxidase. Controls show a low level of S-thiolation, which is increased 3.6-fold during post-ischemic reperfusion. S-Thiolated proteins were purified using streptavidin-agarose, and Western immunoblotting showed HSP27 was present. We increased protein S-thiolation 10-fold with 10 µM H2O2 with or without a kinase inhibitor mixture (staurosporine, genistein, bisindolylmaleimide, SB203580, and PD98059). H2O2 alone induced the phosphorylation of HSP27 Ser-86 and Ser-45/Ser-59 of its homologue alpha B crystallin. However, kinase inhibition reduced phosphorylation of these sites below basal. Despite effective kinase inhibition, H2O2 still disaggregated HSP27, but not alpha B crystallin. This is consistent with the lack of an S-thiolation site on alpha B crystallin. Thus, we have demonstrated a novel mechanism of HSP27 multimeric size regulation. S-Thiolation must occur at Cys-141, the only cysteine in rat HSP27.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Myocardial ischemia and reperfusion is a multifaceted stress, a component of which is an oxidative burden. The oxidizing environment present within the myocardium during ischemia and reperfusion has the dual effects of causing damage (1), as well as initiating stress-adaptive or -protective responses such as ischemic preconditioning (2, 3). S-Thiolation is an oxidative, reversible post-translational modification of cysteine residues of proteins and can be viewed as a protective mechanism that guards against the terminal or irreversible oxidation of these residues.

Protein thiol oxidation involving S-thiolation has all of the essential elements of a regulatory system, including sensitivity, specificity, and reversibility. Thus, S-thiolation may regulate protein function in the same way as other post-translational modifications such as phosphorylation. Protein S-thiolation can be directly coupled to cellular redox status and has no absolute requirement for specialized regulatory enzymes, although thiol transferase enzymes can catalyze these reactions (4). There is increasing evidence that cysteine-targeted oxidation, such as S-thiolation, functions as a major, reversible post-translational regulatory mechanism of many classes of proteins, including structural proteins (5, 6), metabolic enzymes (7-9), ion translocators (10), membrane receptors (11), kinases (12, 13), posphatases (14), transcription factors (15), G-proteins (16, 17), DNA isomerases (18), and proteosome complexes (19). Regulatory cysteine residues are often localized to regions of proteins with a high pKa, as the alkaline pH in these areas promotes the formation of the highly reactive thiolate ion. These particularly reactive, and often catalytically essential, cysteine residues are highly sensitive to a number of cellular oxidants, including glutathione disulfide, cysteine, nitric oxide, nitrosothiols, peroxynitrite, hypochlorous acid, molecular oxygen, and reactive lipids (20-28). The exact reactants involved in the formation of S-thiolated proteins is unclear and may be dependent on a number of specific criteria, including the tissue type, the nature of the oxidative stress and its duration. Potential mechanisms of protein S-thiolation include disulfide exchange with low molecular weight mixed disulfides, such as GSSG or cystine. Stress leading to nitric oxide generation can also promote protein S-thiolation, as nitrosothiols can be formed, which have the dual ability to S-thiolate and S-nitrosylate proteins. Hydrogen peroxide can promote S-thiolation of proteins through activation of thiol groups, involving the formation of sulfenic acid intermediates. Other potential mechanisms of protein S-thiolation involve metal ion catalysis or interaction of protein and small molecule thiyl radicals.

We have used methods that allowed the identification of proteins, which are susceptible to S-thiolation (29), and found that the small stress protein HSP27 is a target for this form of oxidative modification. Under basal conditions, HSP27 exists as a high molecular weight aggregate that is broken down following phosphorylation by stress-activated kinase pathways such as p38MK, which activates MAPKAPK2, which directly phosphorylates HSP27 (30). Other classes of kinase, such as cGMP-dependent protein kinase or casein kinase, are also able to phosphorylate HSP27 (31). The phosphorylation-induced breakdown of the macromolecular structure correlates with the loss of molecular chaperone activity (32, 33). alpha B crystallin shares considerable homology with HSP27, and its multimeric aggregate size is also regulated by phosphorylation (34, 35). However, rat alpha B crystallin differs notably from HSP27 in that it does not contain a single cysteine residue. Consequently, we have used the non-thiolatable alpha B crystallin protein to assess any differential behavior between the two homologues during oxidative stress. In this study we demonstrate that myocardial oxidant stress induces HSP27 S-thiolation and that this process results in the breakdown of the macromolecular complex independently of phosphorylation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- These were obtained from Sigma or BDH unless stated and were of AnalaR grade or above.

Biotinylation of Cysteine-- 120 mg of the water-soluble biotinylation reagent sulfosuccinimidyl-6-(biotinamido)hexanoate was added to 29 mg of cysteine in 2 ml of 10× phosphate-buffered saline and left to derivatize for 1 h at room temperature. The biotin-cysteine was then purified using a Shimadzu HPLC system with an automated fraction collector on an APEX, 5-µm ODS column (Jones Chromatography, Hengoed, UK) with detection at lambda max (190-400 nm) using a diode array detector. The biotin-cysteine was added to the bicarbonate perfusion buffers when required and the concentration confirmed spectrophotometrically using the Ellman reagent (Pierce and Warriner) with cysteine as a standard.

Animals-- Male Wistar rats (200-250 g) were used throughout this study and were obtained from BK Universal. The animals were maintained humanely in compliance with the "Principles of Laboratory Animal Care" formulated by the "National Society for Medical Research" and "Guide for Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication no. 85-23, revised 1985).

Isolated Heart Preparations-- Animals were anesthetized with sodium pentobarbitone (40 mg intraperitoneal) and injected with sodium heparin (200 IU) via the femoral vein. Hearts were rapidly excised, placed in cold (4 °C) bicarbonate buffer, and cannulated. The hearts were then perfused with bicarbonate buffer gassed with 95% O2, 5% CO2 at 37 °C. Perfusion was in the non-recirculating Langendorff mode at a constant flow of 12 ml/g of tissue/min. The bicarbonate buffer contained (in mM) NaCl, 118.5; KCl, 3.1; KH2PO4, 1.18; NaHCO3, 25.0; MgCl2, 1.2; CaCl2, 1.4; and glucose, 10.0.

Perfusion Protocols-- The perfusion protocols used in this study are summarized in Fig. 1. When biotin-cysteine or the antioxidant mercaptopropionylglycine (MPG)1 were used, they were both made up in bicarbonate buffer, immediately before use, to a concentration of 0.5 and 5 mM, respectively. The utility of biotin-cysteine in the investigation of protein S-thiolation has been established, and we have previously shown that it crosses the plasma membrane, gaining access to cytoplasmic and cytoskeletal proteins (29). A kinase inhibitor mixture was used in some protocols and comprised staurosporine (100 nM), genistein (50 µM), bisindolylmaleimide (10 µM), SB203580 (10 µM), and PD98059 (10 µM). Hydrogen peroxide was used a concentration of 10 µM.


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  		Fig. 1.   Isolated rat heart perfusion protocols used in these studies. Biotin-cysteine (0.5 mM) and MPG (5 mM) were made up in bicarbonate buffer immediately before use. The kinase inhibitor mixture was comprised of staurosporine (100 nM), genistein (50 µM), bisindolylmaleimide (10 µM), SB203580 (10 µM), and PD98059 (10 µM). Hydrogen peroxide (10 µM) was used at a concentration of 10 µM. At the end of the protocol, hearts were frozen in liquid nitrogen until further analysis.

Protein Analysis-- Ventricular tissue was homogenized (10 ml of buffer/g of cardiac tissue) on ice in 100 mM Tris-HCl, 5 mM EGTA, 5 mM EDTA, benzamidine (10 µg/ml), leupeptin (100 ng/ml), aprotinin (100 ng/ml), pH 7.0, using a polytron tissue grinder. A sample of the homogenate was reconstituted in sodium dodecyl sulfate (SDS) buffer without a reducing agent. SDS-polyacrylamide gel electrophoresis (PAGE) was carried out using the Bio-Rad mini protean II system. In some samples, to confirm that S-thiolated/biotinylated proteins were modified via disulfide formation, 20 mM DTT was added to the SDS buffer. After electrophoresis samples were transferred to PVDF using a Pharmacia system semi-dry blotter. S-Thiolated proteins were identified by virtue of their biotin tag using streptavidin-HRP (Amersham Biosciences) and the enhanced chemiluminescence reagent (ECL) (Amersham Biosciences, Amersham, UK). PVDF membranes were stained with Coomassie Blue to confirm blotting integrity. Western blots were digitized using a flat-bed scanner (HP Scanjet 11C). The digitized image was then quantitatively analyzed for total proteins S-thiolation in each lane using the NIH-Image software (Freeware, National Institutes of Health, Baltimore, MD).

Antibodies-- The HSP27 and the alpha B crystallin antibodies were supplied by Stressgen Biotechnologies Ltd. (distributed by Bioquote Ltd., York, UK). The HSP27 phosphospecific mouse rabbit polyclonal antibody to rat HSP27 Ser-86 (equivalent to Ser-82 of human sequence) was purchased from New England Biolabs (Hitchin, UK). The alpha B crystallin phosphospecific rabbit polyclonal antibodies (to phosho-Ser-45 and phospho-Ser-59) were kindly supplied by Dr. Kanefusa Kato (34, 35). HRP-linked secondary antibodies were supplied by Amersham Biosciences.

Purification of S-Thiolated Proteins-- S-Thiolated proteins were affinity-purified using streptavidin-agarose. Hearts were homogenized (10 ml of buffer/g of cardiac tissue) on ice in 100 mM Tris-HCl, 5 mM EGTA, 5 mM EDTA, benzamidine (10 µg/ml), leupeptin (100 ng/ml), aprotinin (100 ng/ml), pH 7.0, using a polytron tissue grinder. The homogenate was then centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant (cytosol) was removed and rotated at 4 °C overnight with streptavidin-agarose. The agarose pellet was then extensively washed with phosphate-buffered saline and 1% Triton X-100. S-Thiolated proteins were released from the streptavidin-agarose by treatment with 20 mM DTT in phosphate-buffered saline.

Identification of HSP27 as an S-Thiolated Protein-- Affinity-purified proteins were reconstituted in SDS sample buffer and resolved by SDS-PAGE. After transfer to PVDF membrane, we tested for the presence of HSP27 using an antibody provided by Stressgen Biotechnologies Ltd. (distributed by Bioquote Ltd.).

Effect of GSSG on HSP27 Aggregate Size-- A cardiac homogenate was prepared from isolated rat heart preparations and subjected to 20-min aerobic perfusion, as described above. The homogenate was supplemented with a kinase inhibitor mixture containing: staurosporine (100 nM), genistein (50 µM), bisindolylmaleimide (10 µM), SB203580 (10 µM), and PD98059 (10 µM). Soluble protein was collected by centrifugation for 10 min at 20,000 × g at 4 °C. The soluble protein was then incubated at 37 °C for 5 min with or without 1 mM GSSG, after which it was subjected to gel filtration chromatography.

Gel Filtration-- A Bio-Rad liquid chromatograph with automated fraction collection and detection at 280 nm was used. Cytosolic protein (prepared as described above) was injected on to a Sephracyl gel filtration column (Amersham Biosciences) conditioned with phosphate-buffered saline, pH 7.4, pumped at a flow rate of 1 ml/min via a Shimadzu chromatograph utilizing diode array detection. Fractions were collected by the automated collector, and using immunoblotting, each sample was assayed for the presence of HSP27 or alpha B crystallin. A calibration line was constructed using thyroglobulin (669KDa), catalase (232 kDa), albumin (67 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa) as described in the Amersham Biosciences technical notes.

Statistics-- Results are presented as mean ± S.E. Differences between groups were assessed using analysis of variance followed by a Bonferroni t test. Differences were considered significant at the 95% confidence level.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fig. 2A shows a Western blot probed with streptavidin-HRP, which allows the detection of proteins that are S-thiolated by biotin-cysteine during oxidative stress. Oxidative stress, during post-ischemic reperfusion or hydrogen peroxide treatment, efficiently induced protein S-thiolation in the intact isolated rat heart. Ischemia and reperfusion or hydrogen peroxide treatment both significantly (p < 0.05) increased protein S-thiolation by 3.6- and 10-fold, respectively. Treatment of the samples containing S-thiolated proteins induced by reperfusion or hydrogen peroxide with DTT abolished detection of this oxidative modification. This demonstrates the signals are detected as a consequence of a disulfide bond linking the biotin-cysteine to a protein. Fig. 2B shows a Coomassie-stained SDS-PAGE gel of affinity-purified cytosolic S-thiolated proteins from a heart subjected to ischemia and reperfusion. Approximately 20 dominant S-thiolation substrates have been detected in the cytosol of rat heart, with many more minor substrates present. Presumably, there are other S-thiolation substrates present that are below the detection sensitivity of Coomassie Blue dye. Fig. 2C shows a Western immunoblot of purified S-thiolated proteins probed with an anti-HSP27 antibody and demonstrates that HSP27 was S-thiolated during cardiac reperfusion. Supplementation of hearts with MPG prior to the onset of ischemia decreased the amount of HSP27 that could be purified using streptavidin-agarose to control levels.


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  		Fig. 2.   A, Western blot probed with streptavidin-agarose showing protein S-thiolation during oxidative stress in the isolated rat heart. Ischemia and reperfusion or hydrogen peroxide treatment significantly (p < 0.05) increased protein S-thiolation by 3.6- and 10-fold, respectively. Treatment of the samples with DTT abolished the detection of S-thiolated proteins. B, Coomassie-stained SDS-PAGE gel of affinity-purified S-thiolated proteins from heart subjected to the ischemia and reperfusion protocol shown in Fig. 1. C, Western immunoblot of streptavidin-agarose affinity-purified S-thiolated proteins probed with anti-HSP27. This demonstrates that HSP27 was S-thiolated during cardiac reperfusion. Loading the heart with the thiol antioxidant MPG prior to the onset of ischemia significantly reduced the amount of HSP27 present following affinity purification of the S-thiolated proteins. In effect, this means loading hearts with MPG prior to ischemia reduced HSP27 S-thiolation during reperfusion.

Fig. 3 shows Western immunoblots (using phosphospecific antibodies) and their quantitation. There is a low basal level of HSP27 Ser-86 and alpha B crystallin Ser-45/Ser-59 phosphorylation under control aerobic conditions. Hydrogen peroxide treatment significantly increased the phosphorylation state of each of these residues. The effect of the kinase inhibitor mixture when administered during aerobic perfusion or during hydrogen peroxide treatment was to reduce phosphorylation at all three residues below that detected basally. Thus, the kinase inhibitor mixture efficiently blocked the hydrogen peroxide-induced phosphorylation below that present in aerobically perfused control hearts.


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  		Fig. 3.   Western immunoblots with phosphospecific antibodies to small stress proteins, demonstrating that there is a basal level of HSP27 phosphorylation at Ser-86 and alpha B crystallin at both Ser-45 and Ser-59. Hydrogen peroxide treatment significantly increases the phosphorylation of each of these residues. The effect of the kinase inhibitor mixture when administered during aerobic perfusion or during hydrogen peroxide treatment was to reduce phosphorylation at all three residues below that detected basally.

Fig. 4A shows Western immunoblots (probed with an anti-HSP27 or anti-alpha B crystallin) of fractions collected when rat heart homogenate was separated by molecular weight using gel filtration liquid chromatography. Clearly treatment of the cytosolic fraction with 1 mM GSSG in vitro caused the bulk of the HSP27 to elute from the column in a later fraction, indicating breakdown of the HSP27 multimeric complex. In contrast, GSSG treatment had no effect on the complex size of alpha B crystallin. In these experiments a kinase inhibitor mixture was in the homogenates, and therefore no changes in the phosphorylation state of protein would have occurred in control or GSSG-treated samples.


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  		Fig. 4.   A, Western immunoblots from cardiac homogenates resolved by calibrated gel filtration liquid chromatography. 1 mM GSSG treatment of homogenates in vitro induced breakdown of the macromolecular HSP27 complex in the presence of a kinase inhibitor mixture. In contrast, GSSG had no effect on the complex size of the HSP27 homologue alpha B crystallin. B, Western immunoblots of fractions from cardiac homogenates separated by gel filtration liquid chromatography. Hydrogen peroxide treatment of the isolated rat heart (in the presence of a kinase inhibitor mixture) during aerobic perfusion induced breakdown of the HSP27 macromolecular complex. In contrast and in the same samples, the multimeric size of alpha B crystallin was unaffected by the application of hydrogen peroxide. These observations are consistent with S-thiolation-induced breakdown of HSP27 independently of phosphorylation.

Fig. 4B shows Western immunoblots (probed with an anti-HSP27 or anti-alpha B crystallin) of fractions collected when rat heart homogenates (all of which were perfused with kinase inhibitors) were separated by gel filtration liquid chromatography. Hydrogen peroxide efficiently disaggregated HSP27 despite the presence of kinase inhibitors. In contrast, the kinase inhibitor mixture blocked the hydrogen peroxide-induced breakdown of alpha B crystallin. Hydrogen peroxide-induced breakdown was not due to phosphorylation, as kinase inhibition efficiently blocked hydrogen peroxide-induced small stress protein phosphorylation (see Fig. 3).

Fig. 5 quantifies the GSSG-induced redistribution of HSP27 to a lower molecular aggregate shown by Western immunoblotting in Fig. 4A. It is evident that there was no equivalent breakdown of the alpha B crystallin complex. Quantitation was achieved by designating the total immunoblot signal from all fractions as 100% and then determining what proportion was in each fraction. This quantitative approach confirmed the qualitative assessment of the Western immunoblotting.


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  		Fig. 5.   Quantitative analysis of Western immunoblot data shown in Fig. 4A. Quantitation involved designating the total immunoblot signal from all fractions as 100% and then determining what proportion was in each fraction. In vitro treatment of homogenates changed the molecular size distribution of HSP27, such that there was a large decrease in the size of the aggregate complex. In contrast, GSSG treatment did not initiate a breakdown of the alpha B crystallin macromolecular complex. This differential behavior is consistent with GSSG-induced S-thiolation of HSP27, resulting in breakdown of the multimeric structure.

Fig. 6A is a quantitative analysis of Western immunoblots assessing the breakdown of HSP27 in isolated hearts during aerobic or hydrogen peroxide treatment, in the presence or absence of kinase inhibitors. Quantitation involved designating the total immunoblot signal from all fractions as 100%, after which the relative proportion in each fraction was determined. Clearly, hydrogen peroxide treatment reduced the size of the HSP27 complex, and this occurred regardless of the presence of the kinase inhibitor mixture. Fig. 6B shows quantitatively that hydrogen peroxide also reduced the aggregate size of alpha B crystallin. However, and in contrast to HSP27, this breakdown of the complex was fully inhibited by the kinase inhibitor mixture. The kinase inhibitors had little effect on the basal size of the two multimeric complexes observed under control aerobic conditions; probably reflecting the fact that they are predominantly at their maximal size under control conditions. The fact that hydrogen peroxide-induced disaggregation of HSP27 cannot be blocked by kinase inhibition supports the contention that this is achieved by S-thiolation. This is further supported by the observation that hydrogen peroxide-induced alpha B crystallin breakdown can be fully blocked by kinase inhibition, consistent with the fact that this protein cannot be S-thiolated as it contains no cysteine residues.


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  		Fig. 6.   A, quantitative analysis of HSP27 multimeric size during control aerobic perfusion or hydrogen peroxide treatment (with or without kinase inhibition). Quantitation involved designating the total immunoblot signal from all fractions as 100%, after which the relative proportion in each fraction was determined. Clearly, hydrogen peroxide treatment reduced the size of the HSP27 complex, regardless of the presence of the kinase inhibitor mixture. B, quantitative analysis of alpha B crystallin multimeric size during control aerobic perfusion or hydrogen peroxide treatment (with or without kinase inhibition). Hydrogen peroxide treatment also efficiently reduced the size of the alpha B crystallin complex. However, and in contrast to HSP27, this breakdown of the complex was fully inhibited by the kinase inhibitor mixture. The kinase inhibitors had little effect on the basal size of the two multimeric complexes observed under control aerobic conditions. The fact that the hydrogen peroxide-induced disaggregation of HSP27 cannot be blocked by kinase inhibition supports the contention that this is achieved by S-thiolation. This is further supported by the observation that hydrogen peroxide-induced alpha B crystallin breakdown can be fully blocked by kinase inhibition, consistent with the fact that this protein cannot be S-thiolated as it contains no cysteine residues.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biotin-Cysteine as a Probe for Proteins That Are S-Thiolated-- The study of protein S-thiolation in vivo is technically challenging, and progress in this area has been slow because of the difficulties in assessing this oxidative post-translational modification. Most studies of protein S-thiolation during oxidant stress have used cultured cells with a radiolabeled thiol pool in which the S-thiolated proteins are detected by autoradiography of non-reducing SDS-PAGE gels (36). Alternatively, non-reducing isoelectric focusing gel electrophoresis coupled with Western immunoblotting can be used if the identity of the protein is known, as S-thiolation alters the pI of proteins. In the present study, biotinylated cysteine (biotin-cysteine) has proved extremely useful in the investigation of protein S-thiolation. The presence of a biotin tag on proteins, which become S-thiolated, allowed a range of investigative procedures to be carried out which exploit the high affinity of biotin for avidin derivatives. Thus, S-thiolated proteins were detected on non-reducing Western blots using streptavidin-HRP, and these were quantified via digitization. S-Thiolated proteins were purified using a streptavidin affinity matrix, then identified using Western immunoblotting.

Cardiac Protein S-Thiolation during Oxidative Stress-- Ischemia and reperfusion, as well as hydrogen peroxide treatment, induced S-thiolation of many cardiac proteins. S-Thiolation of proteins during ischemia and reperfusion is particularly noteworthy, as this is a physiologically relevant oxidant stress, whereas many studies use chemical oxidants that cause a physiologically irrelevant redox change. In this connection, the concentration of hydrogen peroxide (10 µM) that we have used throughout this study is also physiologically pertinent.

HSP27 Is a Target for S-Thiolation-- We have shown that HSP27 is S-thiolated during cardiac oxidative stress, and this induces the multimeric aggregate, basal form of this protein to disassemble. S-Thiolation of rat heart HSP27 must have occurred at cysteine 141, as this is the only cysteine residue in this protein. HSP27 from some species, such as human, has a second cysteine toward the C terminus which may also be a target for S-thiolation. It is interesting that the HSP27 homologue alpha B crystallin contains no cysteine residues. This is consistent with our observation that oxidant stress (either by GSSG treatment of homogenates or hydrogen peroxide treatment of isolated rat hearts) in the presence of effective kinase inhibition was unable to disaggregate alpha B crystallin. HSP27 S-thiolation has been previously observed in an in vitro system in which GSSG was added to purified protein. These studies also showed that HSP27 form disulfide-linked dimers and that these paired protein molecules assemble into the large multimeric complexes (37, 38). It has also been shown in a cell model that there is a direct relationship between the multimeric aggregate size of HSP27 and the concentration of GSH, as well as evidence of an interplay between HSP27 and GSH, which regulates the cellular levels of these molecules (32, 39). The oligomeric state of HSP27 regulates its ability to act as a molecular chaperone and could also control the ability of this protein to inhibit the polymerization of actin, an intermediate filament protein, which itself is interestingly also a target of S-thiolation (5, 6). Indeed, in this system we have been able to identify actin as a substrate for S-thiolation during cardiac reperfusion (29).

Modification of HSP27 by Other Oxidants-- The proteins that were S-thiolated during reperfusion are those with reactive cysteines, which may be particularly susceptible to a range of oxidative modification, not just S-thiolation. The actual mode of oxidation may involve modification by a range of species, including glutathione, cysteine, homocysteine, nitrosoglutathione, glutathione sulfonamide, glutathione disulfide S-oxide, nitric oxide, hypochlorous acid, or other oxidizing species (21-27). The cysteines within the G-protein Ha-Ras can be both S-thiolated or S-nitrosylated (17). The type of oxidative cysteine modification which takes place during an oxidative insult will depend on many factors, including the nature, complexity, and intensity of the prevailing oxidant stress.

Redox Modification of Stress Proteins-- HSP33 has previously been shown to be regulated by oxidation of its cysteine residues (40). The chaperone function of this protein is activated by oxidant stress, and the molecular redox switch for this activity change is the formation of a disulfide bond. Transcriptional regulation of HSP27 and alpha B crystallin may also be directly coupled to the cellular redox status (39, 41).

Lens HSPs and Oxidative Stress-- alpha crystallins constitute the major protein component of mammalian lens and help maintain the transparency of this tissue by the action of its chaperone activity, which limits aggregation of proteins during stress (42, 43). During aging or following pathogenic oxidative stress, cataracts form in the lens, and this is associated with damage to proteins and formation of high molecular weight protein complexes consisting of aggregated proteins, including the crystallins (44). It is possible that the crystallins have a role in limiting these damaging processes and so combat the consequences of oxidative stress (45). However, alpha B crystallin knock-out studies have shown that this protein is not directly essential for the development of lens transparency, which contrasts with the crucial role played by its homologue alpha A crystallin (46). alpha B crystallin may have a maintenance role, helping to conserve transparency in an oxidative environment. However, in knock-out studies it is difficult to rule out compensatory actions by up-regulation of homologous proteins. Presumably the normal lens invests in significant amounts of alpha B crystallin because it is beneficial, although it appears non-essential for the development of transparency.

It is noteworthy that rat alpha B crystallin contains no cysteine residues, whereas related proteins such as HSP27 and alpha A crystallin have these thiol-containing amino acids. The reactivity of the thiol groups renders proteins susceptible to oxidation by molecular oxygen, low molecular weight mixed disulfides, nitric oxide, nitrosothiols, reactive lipid species, and other electrophilic species (20-27). Disulfides between cysteine-containing proteins can also be formed during oxidative stress. The fact that alpha B crystallin contains no cysteines renders it immune to such oxidative post-translational modifications. The absence of cysteines in alpha B crystallin may represent a specific biological mechanism that has evolved to allow functional integrity to be maintained in the face of oxidative stress. This contention is supported by the fact that alpha B crystallin is found in abundance in lens and muscle tissue, all of which endure a considerable oxidative burden. To combat this oxidative burden, these tissues maintain high basal levels of antioxidants, particularly the thiol-containing compound glutathione, which help maintain reducing conditions (47, 48).

S-Thiolation of HSP27 Regulates Multimeric Aggregate Size Independently of Phosphorylation-- HSP27 multimers are disassembled in response to phosphorylation, which may be triggered by stress. This is substantiated by studies in which HSP27 was engineered to have its phosphorylation sites replaced with neutral amino acids or amino acids with negative charges, to simulate phosphorylation. Pseudo-phosphorylated HSP27 had a substantially lower multimeric aggregate weight compared with control, demonstrating a profound effect of phosphorylation on the assembly of this protein. Using a mixture of kinase inhibitors, we have been able to block the hydrogen peroxide-induced phosphorylation of HSP27. This blockade was so efficient that the phosphorylation levels were reduced below those found during aerobic perfusion. Despite the blockade of phosphorylation, oxidant stress was able to cause the disaggregation of HSP27.

Biological Relevance of Decreased HSP27 Multmeric Size during Oxidative Stress-- It is clear that oxidative stress results in the breakdown of the multimeric structure of HSP27, and this can be achieved by via phosphorylation (particularly of Ser-86) or S-thiolation at Cys-141 in rat tissues. Breakdown of the multimeric complex will result in the loss of molecular chaperone activity (32, 33). Teleologically, this would appear to be a counter-intuitive stress response. Nevertheless, two independent mechanisms (phosphorylation and S-thiolation) ensure breakdown occurs, implicating breakdown as beneficial process during cellular stress. It is therefore possible that HSP27 may have multiple functions. Under basal, non-stressful conditions it functions as a chaperone, aiding protein folding. During stress, such as ischemia, HSP27 complexes are phosphorylated, causing breakdown, and at the same time this small stress protein also becomes detergent-insoluble (translocation). Translocation seems to be dependent on acidification (which occurs during ischemia) and involves binding to intermediate filament proteins such as actin or desmin, although binding to membrane proteins may also occur (49, 50). We hypothesize that binding of HSP27 to its target proteins may represent a mechanism of protection independent of chaperone activity. The intermediate filament protein desmin and the myofilament protein troponin I are recognized targets for calcium-dependent proteolysis or cross-linking to other proteins, by the enzyme transglutaminase, during ischemia and reperfusion (51-54). The fact that HSP27 binds to filamentous proteins may allow it to interfere with these injurious events and provide protection. Indeed, it was recently shown that troponin I proteolysis is attenuated by ischemic preconditioning, a mechanism that enhances the translocation of small stress proteins (55, 56). HSP27 not only binds to intermediate filaments such as actin during cellular stress, but it is also well established that it regulates the polymer dynamics of these structures (57-59). In this way, HSP27 can regulate the cytoskeletal cell strength, and this could play an important role in providing resistance or adaptation to stress. For example, swelling-induced damage is a pivotal mechanism of injury during ischemia and reperfusion (60, 61); thus, HSP27-mediated cell strengthening in ischemia/reperfusion would be a potential mechanism of endogenous cardioprotection. This would be consistent with a number of cellular studies showing that small HSPs provide protection against simulated ischemia involving a hypotonic stress that causes swelling-induced damage (57, 62-66).

It is clear that both phosphorylation and S-thiolation regulate the multimeric aggregate size of HSP27. It is not clear what the functional significance of these dual mechanisms of disaggregation is or whether there is a synergistic interaction, which may be important for reasons other than decreasing aggregate size. Clearly, there are multiple sites of HSP27 phosphorylation, and they do not all appear to be required for complex breakdown. Consequently, we hypothesize that HSP27 phosphorylation (especially at residues other than Ser-86) may have a role in targeting protein binding during stress-induced translocation. It is possible that phosphorylation increases the affinity of the disaggregated HSP27 for specific target proteins.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Cardiac Physiology, The Centre for Cardiovascular Biology and Medicine, The Rayne Inst., St Thomas' Hospital, London SE1 7EH, UK. Tel.: 44-20-7928-9292 (ext. 2749); Fax: 44-20-7922-8139; E-mail: philip.eaton@kcl.ac.uk.

Published, JBC Papers in Press, March 29, 2002, DOI 10.1074/jbc.M200591200

    ABBREVIATIONS

The abbreviations used are: MPG, mercaptopropionylglycine; DTT, dithiothreitol; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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