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The FASEB Journal • Research Communication

Senile graying: H2O2-mediated oxidative stress affects hair color by blunting methionine sulfoxide repair

J. M. Wood,*,†,1 H. Decker,‡ H. Hartmann,‡ B. Chavan,* H. Rokos,*,† ʈ J. D. Spencer,*,† S. Hasse,*,† M. J. Thornton,* M. Shalbaf,* R. Paus,§, and K. U. Schallreuter*,2 *Department of Biomedical Sciences, Clinical and Experimental Dermatology, and †Institute for Pigmentary Disorders, University of Bradford, Bradford, UK; ‡Institute of Molecular Biophysics, University of Mainz, Mainz, Germany; §Department of Dermatology, University of Lu¨beck, Lu¨beck, ʈ Germany; and University of Manchester, Manchester, UK

ABSTRACT Senile graying of human hair has been decline—especially in today’s world, where are the subject of intense research since ancient times. confronted with increasing pressure to stay “forever Reactive oxygen species have been implicated in hair young and vital.” Hence, senile and premature graying follicle apoptosis and DNA damage. Here has long attracted researchers and industry alike with we show for the first time by FT-Raman spectroscopy in scientific as well as commercial targets. Yet, apart from vivo that human gray/white scalp hair shafts accumu- various hair dyes of varying efficacy and duration, fully late (H2O2) in millimolar concen- satisfactory solutions for the graying problem remain to trations. Moreover, we demonstrate almost absent cat- be brought to market. A key reason still not addressed alase and methionine sulfoxide reductase A and B is that the underlying molecular and cellular mecha- protein expression via immunofluorescence and West- nisms of graying remain under debate (1–3). ern blot in association with a functional loss of methi- So far, the biological process of hair graying has been -O) repair in the entire gray attributed to the loss of the pigment-forming melano؍onine sulfoxide (Met-S O formation of Met cytes from the aging , including the bulb؍hair follicle. Accordingly, Met-S residues, including Met 374 in the active site of tyrosi- and the outer root sheath (1, 2, 4–12). In this context, nase, the key enzyme in melanogenesis, limits enzyme it is of interest that activity of hair bulb is functionality, as evidenced by FT-Raman spectroscopy, under cyclic control (1, 13). Both melanogenesis and computer simulation, and enzyme kinetics, which leads hair shaft production take place in the anagen phase of to gradual loss of hair color. Notably, under in vitro the hair cycle. Toward the end of this phase, the conditions, Met oxidation can be prevented by L- pigment-forming melanocytes retract their dendrites methionine. In summary, our data feed the long-voiced, and stop melanogenesis, which is followed by apoptosis- but insufficiently proven, concept of H2O2-induced driven regression in the catagen phase and the final oxidative damage in the entire human hair follicle, resting telogen phase. Theories for the gradual loss of inclusive of the hair shaft, as a key element in senile pigmentation include exhaustion of enzymes involved hair graying, which does not exclusively affect follicle in melanogenesis, impaired DNA repair, loss of telom- melanocytes. This new insight could open new strat- erase, antioxidant mechanisms, and antiapoptotic sig- egies for intervention and reversal of the hair graying nals including the loss of Bcl-2 and decreased process.—Wood, J. M., Decker, H., Hartmann, H., factor (2, 4, 6, 14–17). Vacuolation in hair bulb mela- Chavan, B., Rokos, H., Spencer, J. D., Hasse, S., nocytes has been ascribed to oxidative stress (18). By Thornton, M. J., Shalbaf, M., Paus, R., Schallreuter, analogy with the free radical theory of aging, recently a K. U. Senile hair graying: H2O2-mediated oxidative “free radical theory of graying” has been proposed (4). stress affects human hair color by blunting methio- Generation of H2O2 has been attributed to its intrinsic nine sulfoxide repair. FASEB J. 23, 2065–2075 (2009) release from the melanogenesis pathway, ultimately yielding apoptosis of hair follicle melanocytes (HFMs) Key Words: MSRA&B ⅐ tyrosinase ⅐ ⅐ follicle cells and DNA damage (4). Decline of melanogenesis is

1 and hair color have been at the center of Deceased. 2 Correspondence: Clinical and Experimental Dermatol- attention of humans since ancient times. Graying of the ogy/Department of Biomedical Sciences, University of Brad- hair (canities) has major psychosocial and socioeco- ford, Bradford, BD7 1DP, West Yorkshire, UK. E-mail: nomic implications, since it is often regarded as a sign [email protected] of rapidly progressing , ill health, and bodily doi: 10.1096/fj.08-125435

0892-6638/09/0023-2065 © FASEB 2065 associated with loss of tyrosinase (EC 1.14.18.1) activity oxidation products in the native senile human gray/ (2, 14, 19, 20), which affects in turn the rate-limiting white anagen hair shaft. We also show for the first time step in melanogenesis. Here it is of interest that low the presence and function of Met-SϭO repair capacity Ͻ ϫ Ϫ3 H2O2 concentrations ( 0.3 10 M) increase tyrosi- by the enzymes MSRA and MSRB in situ and on the Ϫ nase activity, while high concentrations (10 3 M) irre- functional level in human isolated anagen hair follicles versibly deactivate the enzyme (21, 22). Abundant and in isolated different human hair follicle cells. evidence indicates that many proteins and peptides, Notably, protein expression as well as its function is including the H2O2-reducing enzyme catalase (EC nearly completely abrogated in the gray hair follicle. 1.11.1.6) as well as the two repair mechanisms for free Our results are in agreement with the near absence of and bound methionine sulfoxide (Met-SϭO), methio- H2O2-reducing capacity by catalase, which affects the nine sulfoxide reductases A and B (MSRA and MSRB; entire gray hair follicle. In addition, we show for the ϭ EC 1.8.4.6 MSRA&B), are structurally damaged and first time formation of Met-S OinH2O2-oxidized functionally altered by H2O2-mediated oxidation. This tyrosinase by in vitro FT-Raman spectroscopy, and we finding is based on the presence of methionine (Met), prove loss of enzyme function after oxidation. These cysteine (Cys), and tryptophan (Trp) residues in their results are supported in cell extracts from human protein sequence (23–27). Bearing all these facts in epidermal melanocytes (EMCs) and by enzyme kinet- mind, it was tempting to readdress the potential of ics. Computer modeling of the tertiary structure of H2O2 in senile hair graying. By analogy, we turned to H2O2-oxidized tyrosinase reveals enzyme deactivation , a depigmentation disorder, as this model could due to Met 374 instability in the enzyme active site. hold lessons for a better understanding of the graying Notably, H2O2-mediated oxidation of tyrosinase can be process. In this disease, H O -mediated oxidative stress prevented through free l-methionine, which in turn 2 2 ϭ plays a central role in the loss of inherited skin color acts as an H2O2 scavenger via formation of Met-S O. due to generation and accumulation of millimolar H2O2 concentrations in the epidermal compartment, as documented in vivo by Fourier transform Raman MATERIALS AND METHODS spectroscopy (FT-Raman spectroscopy) (11, 28). Given that hair color is exclusively produced in the pigmen- All studies on human material were approved by the local tary unit of the anagen III-VI hair follicle (2, 14, 19), we ethics committees and were in agreement with the Helsinki declaration. postulated that accumulation of H2O2 could be a contributing factor in the hair graying process due to Cell culture of dermal papilla (DP) cells, dermal fibroblasts deactivation of catalase as well as of MSRA and MSRB, (DFs), dermal sheath (DS) cells, and EMCs which in turn would affect the formation of hair color via tyrosinase in HFMs (11, 25, 26, 28). Hair follicle cell cultures were established from face-lift This idea was supported by the fact that precursor surgery (nϭ2 female) and healthy donors (nϭ3 male) after tyrosinase contains 17 Cys, 14 Trp, and 15 Met residues signed consent. The study was in agreement with the princi- in its sequence, according to the Swiss-PROT database ple of the Helsinki declaration and was approved by the local (http://www.expasy.org), while in mature tyrosinase ethics committees. To establish primary cultures of DFs, the (19–529), 16 Cys, 13 Trp, and 14 Met residues are skin samples were dissected through the dermis; divided into 3- ϫ 3-mm pieces; and transferred to a 25-cm2 tissue-culture present, which are all potentially susceptible to oxida- flask (Dow Corning, Corning, NY, USA). The samples were tion. Additional support for this hypothesis arrives from then cultured in DMEM (Life Technologies, Inc., Paisley, a 3-dimensional model of the mammalian tyrosinase Scotland) supplemented with glutamine (10 mM), penicillin active site (29, 30). Homology modeling of the murine (100 U/ml), streptomycin (100 ␮g/ml), and amphotericin B ␮ enzyme and delineation of key amino acids in the active (250 g/ml), with 20% fetal calf serum (FCS) (Sigma, Poole, Dorset, UK) at 37°C in 5% CO in air. To establish primary site of this enzyme with the help of site-directed muta- 2 cultures of DP and DS cells, follicles were tion analyses revealed that Met 374 was essential for dissected individually and cleaned from fat using two 7.5- tyrosinase activity, because mutation of this residue to gauge needles. The upper part of the hair follicle was Gly 374 caused a 93% loss of enzyme activity (29). Here removed. The hair shaft was removed from the lower part of it is noteworthy that 85% of the primary sequences in the hair follicle by applying gentle pressure to the base, mouse and human tyrosinases are conserved, with 92% leaving the DS and the DP. To isolate the DP, a slit was made structural homology. Further support for the critical in the DS, which was inverted by itself to expose the DP on its stalk at the base of the follicle. The DP was separated from the importance of Met 374 stems from studies with human DS by dissecting through its stalk and transferred to a 35-mm type 1 oculocutaneous (OCAI), where the dish containing 2 ml of MEM supplemented with 20% human orientation of this Met residue is affected by the Ser 380 serum. Once the DP cells were isolated, the lower part of the to Pro 380 polymorphism (31, 32). Here this structural DS was transferred to a separate 35-mm dish containing 2 ml change destabilizes Met 374 and disrupts the coordina- DMEM supplemented with 20% human serum. A scratch was tion of His residues to the Cu-(B) active site of tyrosi- made through the tissue to aid attachment to the dish and encourage growth and migration of the DP or DS cells. nase, leading to the loss of enzyme activity (33). Approximately 6 DP or DS cells were cultured in each dish To probe our hypothesis, we applied in vivo FT- and clustered to encourage cell migration. The dishes were Raman spectroscopy, and we show for the first time the left undisturbed for 7 d. After this period, the growth medium presence of millimolar H2O2 concentrations and H2O2 was refreshed, and the explants were checked for cell migra-

2066 Vol. 23 July 2009 The FASEB Journal WOOD ET AL. tion. This step was repeated every 2–3 d until sufficient Western blotting growth had occurred to allow passaging of the cells with resuspension into a T25 flask. Once explant cultures were Gray and hair follicle extracts were obtained as de- established, the medium was supplemented with 10% FCS. scribed above. Normal human EMC and epidermal keratino- ϭ EMCs were cultured from face-lift surgery (n 2) following cyte extracts were used as positive controls and obtained as the method of Pittelkow and Shipley, as described in detail described previously (23, 36, 37). Sample buffer (10% SDS, elsewhere (34). Human HFMs were a generous gift from D. J. mercaptoethanol, glycerol, and 0.5 M Tris/HCl) was added to Tobin (University of Bradford, Bradford, UK). the supernatants before loading on to a 12% polyacrylamide gel for protein separation. The polyacrylamide gel was then Preparation of human anagen hair follicle electroblotted onto a PVDF membrane (Millipore, Billerica, MA, USA) before any nonspecific binding sites were blocked Human hair follicles were isolated from normal human scalp by immersing the membrane in a 0.5% gelatin (Sigma)/ skin obtained from routine face-lift surgery after patient TBS-T buffer (20 mM Tris buffered saline with 0.047% consent (nϭ6; 4 female, 2 male). Anagen VI hair follicles Tween, pH 7.4) blocking solution for2hatroom tempera- were isolated using a microdissection microscope according ture. This step was followed by an overnight incubation at to a modified method of Philpott (35). Briefly, scalp skin was room temperature with the primary antibodies in 0.05% cut in small pieces of ϳ1cm2 in size. The dermis was gelatin/TBS-Tween. The antibodies used were rabbit anti- separated from the subcutis, giving rise to the midlower part MSRA (Labfrontier, Seoul, South Korea; 1:2000), rabbit of anagen VI hair follicles, which were extracted with fine anti-MSRB (Labfrontier; 1:2000), mouse anti-catalase (Sigma; forceps. 1:2000), and goat anti-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:500). Following a wash of 40 min in Preparation of cell extracts from full hair follicles and hair TBS-T buffer, the blot was further incubated for1hatroom follicle cells temperature with an anti-rabbit, mouse, or goat immunoglobu- lin G (IgG) peroxidase-conjugated antibody (Sigma; 1:5000). Visualization of the specific protein bands was performed using To avoid protein denaturation, cell extracts were obtained modified enhanced chemiluminescence (ECL) fixed on a film from intact anagen hair follicles and cell cultures utilizing a sheet (X-OMAT; Kodak, Rochester, NY, USA). minipestle, a mortar (Ϫ80°C), and fine sand. Cell pellets and hair follicles were ground in ice-cold Tris buffer (0.05 M, pH 7.5) followed by centrifugation at 7000 g for 5 min. The Determination of tyrosinase activity supernatant was collected, divided into aliquots, and stored at Ϫ80°C until further use. The protein content was determined Tyrosinase activity was based on the rate of l-dopachrome by the Dc-protein assay (Bio-Rad, Hercules, CA, USA). formation, measured spectrophotometrically at the optical density of 475 nm (Pharmacia, Milton Keynes, UK), when In situ immunofluorescence protein detection in the isolated l- was used as substrate. The reaction mixture con- human hair follicle tained 84 U of mushroom tyrosinase (Sigma) and different l-tyrosine (Sigma) concentrations ranging from 0 to 5 ϫ 10Ϫ3 M in a final reaction volume of 1 ml potassium phosphate Freshly isolated hair follicles were embedded in OCTTM Ϫ buffer (0.1 M, pH 7.0). The rate of reaction was determined compound (Sakura, Eastbourne, UK) and kept at 80°C over the linear period of 2 min. Since 1 ϫ 10Ϫ3 M l-tyrosine until further processing with the cryostat (Leica Microsys- produces 1 ϫ 10Ϫ3 MHO (21), we used 3 ϫ 10Ϫ3 M tems, Milton Keynes, UK). Frozen slides with 5-␮m cryosec- 2 2 ϭ ϭ l-tyrosine (Sigma). The experiments were performed in tions of gray hair (n 3) and dark hair (n 3) follicles and the presence of different l-methionine concentrations (2– shafts were air-dried for1hatroom temperature, fixed in 8ϫ10Ϫ3 M). All experiments were performed in duplicate. ice-cold methanol for 6 min, and blocked in 10% normal Since Met-tyrosinase is activated by l-dopa, we decided to donkey serum (NDS, Jackson Immunoresearch Laboratories, follow l-dopachrome formation from l-dopa at 570 nm. Cambridge, UK) for 90 min, followed by a 5-min wash in l-Dopachrome was determined every 2 min over 15 min using phosphate buffered saline (PBS). MSRA was detected by a microplate reader (Dynex Technologies, Worthing, UK). using a polyclonal rabbit anti-human antibody (Autogen To test the effect of l-methionine as an H2O2 scavenger, we Bioclear, Calne, UK) diluted 1:50 in 1% NDS, followed by tested the capacity of l-methionine to prevent H O -medi- incubation at room temperature for 3 h. MSRB detection 2 2 ated oxidation. Briefly, both l-methionine and H2O2 (1:1, utilized a monoclonal mouse anti-human antibody (Autogen 5:1, 10:1) were incubated for 15 min to allow the formation of Bioclear) diluted 1:50 in 1% NDS and incubated overnight at Met-SϭO. Then this complex was added to the above reac- 4°C. The detection of catalase utilized a monoclonal mouse tion, and measurements were taken every 2 min. H2O2 anti-human antibody (Sigma) diluted 1:100 in 1% NDS and scavenging was also tested in the presence of 10 ϫ 10Ϫ3 M incubated overnight at 4°C. Slides were washed 4ϫ with PBS, l-methionine where only the H2O2 concentrations were air-dried, and incubated for1hatroom temperature with a changed (0.8, 1.6, 3.3, 6.6ϫ10Ϫ3 M). Enzyme rates were fluorescent secondary antibody [fluorescein isothiocyanate determined over the linear reaction period per minute per (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)- milligram tyrosinase. conjugated donkey anti-rabbit or anti-mouse at a dilution of 1:100 (Jackson Immunoresearch Laboratories)] followed by a wash 3ϫ with PBS, air-dried, and mounted in Vectashield Determination of tyrosinase activities in EMC and HFM Mounting Medium (Vector Laboratories, Peterborough, UK) extracts containing DAPI (4Ј,6-diamidino-2-phenylindole) for nuclear identification. Slides were viewed under a Leica DRMIB/E To determine tyrosinase activity in melanocytes we used fluorescence microscope (Leica Microsystems, Wetzlar, Ger- microplates. Briefly, cell extracts (100 ␮l) of two different many), and images were captured using a digital camera primary human EMC lines (nϭ2) and two different pig- (C8484–05G; Hamamatsu Photonics UK, Welwyn Garden mented HFM lines (nϭ2) were placed into a 96-well plate, City, UK) coupled to a computer and evaluated with the and 11 ␮l l-dopa (2ϫ10Ϫ3 M) (Sigma) was added. The imaging software IPLab 3.7.4 (Scanalytics, Fairfax, VA, USA). formation of l-dopachrome was determined at 570 nm every

H2O2-MEDIATED OXIDATIVE STRESS IN GRAYING HAIR 2067 10 min over 90 min using a microplate reader (Dynex package was used, applying force field yasara3. For calcula- Technologies). The product formation was followed by sub- tion, a water-filled cell was used. tracting the blank from each obtained value. The reaction rate was again determined over the linear reaction period per

minute per milligram of protein. To study the effect of H2O2 on tyrosinase, enzyme activity was determined in the presence RESULTS ϫ Ϫ3 (2 10 M) and absence of H2O2 in EMC extracts. Experi- ments were performed in triplicate. Due to limitation of HFM In vivo FT-Raman spectroscopy identifies the presence of ؍ extract, we were unable to repeat this experiment in these millimolar H2O2 concentrations and Met-S O in the senile cells. Reaction rates were determined per minute per milli- gray and white hair shaft gram of protein. FT-Raman spectra show in vivo the presence of 10Ϫ3 MHO Determination of MSRA and MSRB enzyme activities 2 2 concentrations in gray and completely white hair, although

no H2O2 is detectable in pigmented (Fig. 1). Complete hair follicle extracts were prepared from isolated ϭ Ϫ1 H2O2 assignment is based on the O O stretch at 875 cm hair follicles as described above for EMCs/HFMs and other (28). Moreover, we demonstrate oxidation of Met residues to hair follicle cells. Briefly, the reaction mixture contained 50 Met-SϭO (assigned at 1030 cmϪ1), Trp residues to 5-OH-Trp ␮ Ϫ l cell extract, dithiothreitol (DTT) as an electron donor (10 (assigned at 930 cm 1), and N-formyl kynurenine/kynure- ␮ ϫ Ϫ3 14 ␮ Ϫ l, 5 10 M), and [ C]methionine sulfoxide (10 l, nine (assigned at 1050 cm 1) and Cys residues to cysteic acid ϫ Ϫ3 Ϫ 5 10 M) (23, 36). Reactions were incubated for 60 min at (assigned at 1040 cm 1) (24, 38, 39) (Fig. 1). Based on these ␮ room temperature. Then 5 l of the reaction product was data, we can conclude that gray/white hair shows massive applied to a TLC silica gel plate (GF 1000 ␮m; Merck, H2O2 concentrations associated with H2O2-mediated oxida- Darmstadt, Germany) and chromatographed in isopropanol: tion of critical amino acid residues. formic acid:water (20:1:10). l-Methionine and Met-SϭO were detected by ninhydrin. Radiolabeled [14C] l-methionine spots were scraped from the TLC plates and added to 3.0 ml Human hair follicle cells hold the capacity for O repair؍of scintillation fluid (Ready Safe; Beckman Coulter, Fuller- functioning Met-S ton, CA, USA) and counted on the 14C channel in a Packard Tricarb Liquid Scintillation Counter (2001 TR; Packard In- 14 As discussed above, H2O2 oxidizes free and protein- struments, Meriden, CT, USA). The amount of [ C] l- ϭ methionine formed was standardized to micromoles per bound Met to R- and S-Met-S O, which are normally milligram of protein per 30 min. repaired by MSRA and MSRB, respectively (41). How- ever, with old age, both enzyme levels decrease, and, in In vitro and in vivo FT-Raman spectroscopy for detection of the case of H2O2-mediated stress, this important mech- ؍ H2O2, Met-S O, cysteic acid, 5-OH-tryptophan, and anism is susceptible to oxidation by this ROS (36, 42). kynureneic acid in H2O2-oxidized tyrosinase and gray To the best of our knowledge, so far the presence of hair MSRA and MSRB in the human hair follicle has not been documented. Here we show in vitro protein ex- FT-Raman spectra were acquired using a Bruker RFS 100/S pression of catalase, MSRA, and MSRB proteins in spectrometer (Bruker, Karlsruhe, Germany) with a liquid- human HFMs, DP cells, DS cells, and DFs by immuno- nitrogen-cooled germanium detector. Near-infrared excita- tion was produced by a Nd3ϩ:YAG laser operating at 1064 nm. Each spectrum was accumulated over 17 min with 1000 scans Ϫ1 and a resolution of 4 cm . Detection of H2O2 is based on the OϭO stretch at 875 cmϪ1 (28). The Met-SϭO stretch was visualized at 1030 cmϪ1 accordingly (38). Cysteine and its oxidation product cysteic acid is assigned to the SO stretch at 1040 cmϪ1 (39). l-Tryptophan and its oxidation products 5-OH-tryptophan (930 cmϪ1) and N-formyl-kynurenine/ kynurenine (1050 cmϪ1) were recently assigned (24). Native ϫ Ϫ3 and H2O2-oxidized tyrosinase (10 10 M) were lyophi- lized and measured as solids. Native human gray, com- pletely white, and brown/ hair shafts from the scalp hair were cut into small pieces (1 mm) and analyzed by FT-Raman spectroscopy.

Computer modeling of tyrosinase

A 3-D homology model of mTy (Mus musculus, gi:6755913) and hTy (Homo sapiens, gi:401235) was reconstructed based Figure 1. FT-Raman spectroscopy identifies in vivo millimolar

on the crystal structure from a tyrosinase as previously re- amounts of H2O2 and its oxidation products in gray and white ⅷ ported (29). Both mTy and hTy are 98% identical around the hair shafts. Peaks are assigned accordingly: ,H2O2 at 875 ϭ ء Ϫ1 Ϫ1 active site. The alignment and corrections are performed as cm ; §, phenylalanine at 1004 cm ; , Met-S O at 1030 described previously (29). Two diastereomers of oxidized cmϪ1; #, 5-OH Trp at 930 cmϪ1; f N-formylkynurenine/ methionine require a tetrahedral coordination with an open kynurenine at 1050 cmϪ1; ࡗ, cysteic acid at 1040 cmϪ1). coordination. The molecular graphics were generated with Spectrum 1, brown hair; spectrum 2, gray hair; spectrum 3,

Yasara Twinset 7.12.5 (40) and rendered with Povray 3.6 white hair. Note that no evidence for H2O2 or any oxidation (www.povray.org). For MD simulations, the Yasara 7.12.5 products was found in brown hair.

2068 Vol. 23 July 2009 The FASEB Journal WOOD ET AL. fluorescence and Western blot analysis in all cell types blot analysis supports the decreased in situ protein (Fig. 2A). Determination of enzyme function of MSRA expression in the gray anagen hair follicle (Fig. 3B). and MSRB in cell extracts from those cells reveal These data are substantiated further by low MSRA and significantly higher activities in DP cells compared to MSRB enzyme activities in anagen hair follicle extracts DFs and DS cells, while enzyme activities in HFMs are from gray compared to brown hair follicles (Fig. 3C). similar to EMCs, as recently documented (36) (Fig. Compelling evidence in vivo and in vitro indicates that 2B). It is interesting that DP cells have at least under in H2O2-mediated oxidative stress in the entire graying vitro conditions the highest repair capacity. This result anagen hair follicle lowers both catalase and Met-SϭO supports the importance of a very efficient redox repair via MSRA and MSRB, feeding in turn a vicious balance/repair within the central position of the pa- cycle for redox dysbalance. pilla, and it needs further investigation. In vitro FT-Raman spectroscopy confirms formation O formation in tyrosinase after؍Low catalase levels in the gray human hair follicle of Met-S ؍ correlate with decreased Met-S O repair H2O2-mediated oxidation

One major degrading enzyme for H2O2 is the ubiqui- We next turned our interest to tyrosinase, which is tous heme protein catalase. Low concentrations of the the key enzyme for melanogenesis. Considering that enzyme have been shown in old age, including the this enzyme contains several targets for H2O2-medi- aging hair follicle, and in vitiligo (43–45). In this ated oxidation in its sequence, including a crucial context, it is noteworthy that oxidation of catalase by its methionine in position 374 in the active site, we own substrate causes enzyme deactivation (25, 26). In decided to utilize again FT-Raman spectroscopy to situ protein expression of catalase, MSRA, and MSRB follow the oxidation products. The results identified shows greatly reduced levels in the gray hair follicle the presence of Met-SϭO as a fingerprint of oxidized compared to the pigmented follicle (Fig. 3A). Western Met residues, cysteic acid from Cys, and 5-OH-Trp/N-

Figure 2. Human hair follicle cells hold functioning MSRA and MSRB activities. A) In vitro expression of MSRA and MSRB in human hair follicle cells. a) Scheme of the anagen hair follicle, showing location of DP, DS, DFs, and HFMs. b) Immunoreactivity expression of MSRA, MSRB, and catalase in HFMs, DP, DS, and DFs (green, FITC- labeled; , TRITC-labeled). Expression of catalase was

included as internal marker for H2O2-mediated oxidative stress (11). c) Western blot confirms catalase, MSRA, and MSRB expression at the expected size in HFM extracts. B) MSRA and MSRB enzyme activities are present in hair follicle cells. Specific activities are shown in micromoles per milligram of protein per 30 min. DP cells, n ϭ 3; DFs, n ϭ 3; DS, n ϭ 3; HFMs, n ϭ 2; EMCs, n ϭ 2; EKCs (epidermal keratinocytes), n ϭ 2. Note the high expression in DP and HFMs/EMCs, underscoring the importance of Met-SϭO repair in these cells. Error bars ϭϮsem.

H2O2-MEDIATED OXIDATIVE STRESS IN GRAYING HAIR 2069 Figure 3. Reduced MSRA and MSRB protein expression correlates with reduced enzyme function in the gray hair follicle. A) Reduced in situ expression of catalase, MSRA, and MSRB in the entire gray human anagen hair follicle.

Decreased catalase expression is in agreement with H2O2-oxidized enzyme (11). DAPI (blue) indicates nuclei in the corresponding hair follicle (ϫ400). B) Western blot confirms reduced catalase, MSRA, and MSRB in human gray anagen hair follicle extracts. EMCs (EC) and epidermal keratinocytes (KC) served as positive controls (36). C) Decreased MSRA and MSRB activities in human gray anagen hair follicle extracts. Enzyme activities were determined in 3 samples/hair color. Activities are assessed in micromoles per milligram of protein per 30 min. rMSRA/MSRB activity served as positive control. Error bars ϭϮsem.

formylkynurenine/kynurenine from Trp residues in the Met-SϭO. To do so, we decided to follow the l- oxidized enzyme (Fig. 4A). The oxidized enzyme has no dopachrome formation of tyrosinase at 570 nm, activity (Fig. 4B). because l-dopa is a better substrate for tyrosinase under in vitro conditions. We incubated l-Met and H O in a 1:1, 5:1, and 10:1 complex for 15 min and L-Methionine prevents H2O2-induced inhibition of 2 2 tyrosinase added them to a standard reaction after 10 min. The enzyme rates were the same as in the standard Ϫ Ϫ reaction (0.016 min 1 mg protein 1) in the presence Under normal Michaelis-Menten conditions, tyrosi- Ϫ1 Ϫ3 of 5- and 10-fold higher l-Met (0.011 min mg nase is deactivated by 10 M l-tyrosine (22, 46, 47). Ϫ Ϫ Ϫ protein 1, 0.018 min 1 mg protein 1, respectively), During the catalytic cycle from l-tyrosine to while the 1:1 complex was not effective (0.0031 l-dopachrome, both H O and O are stoichiometric Ϫ Ϫ 2 2 Ϫ 2 1 1 byroducts (48, 49). Hence, 10 3 M l-tyrosine yields min mg protein ) to prevent inhibition of the Ϫ3 enzyme. These results indicate that l-Met concentra- 10 MH2O2. These concentrations should be suffi- cient to deactivate the enzyme by oxidizing Met 374 tions need to exceed H2O2 concentrations in order to Met-SϭO, thus explaining tyrosinase suicide inhi- to scavenge this ROS effectively. To support these ϫ Ϫ3 bition by its own substrate. In a first attempt to data further, we used 10 10 M l-Met and ϫ Ϫ3 explore this hypothesis, we used different concentra- different H2O2 concentrations (0.8–6.4 10 M) tions of l-tyrosine as substrate for tyrosinase. The and incubated them with tyrosinase for 10 min. The result from this experiment confirmed our earlier reaction was started with addition of l-dopa. l-Met in this concentration protects the reaction in the pres- finding that the enzyme is inhibited in a concentra- Ϫ Ϫ Ϫ3 ϫ 3 ϫ 3 tion-dependent manner (0.2–5ϫ10 M) (22) (data ence of 0.8 and 1.6 10 M, whereas 3.2 10 M not shown). Next, we followed the reaction in the already exceeds the capacity at this l-Met concentra- Ϫ presence of 3 ϫ 10 3 M l-tyrosine, which would yield tion (Fig. 4D). From these experiments, we can ϫ Ϫ3 3 10 MH2O2. In the presence of different l-Met conclude that the protein-bound Met 374 in the Ϫ3 concentrations (2–8ϫ10 M), the inhibition can be active site of the enzyme is a target of H2O2-mediated prevented in a dose-dependent manner (Fig. 4C). oxidation. Free l-Met is an efficient H2O2 scavenger This result indicated that Met-SϭO formation can be due to formation of free Met-SϭO over the protein- prevented on addition of l-Met. We next tested how bound Met-SϭO formation, if the concentrations are effective l-Met could scavenge H2O2 by forming sufficiently high. This result underlines once more

2070 Vol. 23 July 2009 The FASEB Journal WOOD ET AL. Figure 4. Free l-methionine acts as an effective H2O2 scavenger. A) In vitro FT-Raman spectroscopy confirms the presence of tryptophan to ,(ء)Met-SϭO in oxidized mushroom tyrosinase. Spectrum shows oxidation of methionine residues to Met-SϭO ࡗ 5-OH-tryptophan (#), and cysteine to cysteic acid ( ) with the phenylalanine peak (§). B)H2O2-oxidized tyrosinase has no activity. Enzyme activity was determined in mushroom tyrosinase following the l-dopachrome formation at 475 nm over 10 min ࡗ Œ in the absence of H2O2 ( ) and in the presence of laser-induced H2O2 ( ). This result supports enzyme deactivation due to oxidation of Met 374 in the active site. C) l-Met prevents H2O2-mediated oxidation of tyrosinase in a concentration-dependent Ϫ3 Ϫ3 manner. Given that 10 M l-tyrosine yields 10 MH2O2 (48, 49), we followed tyrosinase activity in the presence of different l-tyrosine concentrations at OD 475 nm (data not shown). Tyrosinase activity is inhibited by 3 ϫ 10Ϫ3 M l-tyrosine (Œ). Inhibition can be prevented by l-Met (0–8ϫ10Ϫ3 M) in a concentration-dependent manner, indicating that addition of l-Met ࡗ ϫ Ϫ3 prevents H2O2-induced oxidation of protein-bound methionine residues in tyrosinase ( ). D) l-Met (10 10 M) prevents ϫ Ϫ3 H2O2-mediated inactivation of tyrosinase. Tyrosinase was incubated for 10 min with l-Met (10 10 M) together with H2O2 (0.8, 1.6, 3.2, 6.4ϫ10Ϫ3 M), and the reaction was started on addition of l-dopa. Reaction rates were determined per minute. ϫ Ϫ3 Result shows that excess of free l-Met protects the enzyme up to 1.6 10 MH2O2, while higher concentrations (3.2 and 6.4ϫ10Ϫ3 M) are too high for the amount of l-Met used in the experiment. Control reaction was carried out without any addition. l-Met alone has no effect on the enzyme reaction (data not shown). All reactions were done in duplicates. the importance of Met 374 in the enzyme active site Computer modeling confirms the loss of functioning (29, 31–33). Met 374 in the active site of human and mouse tyrosinase after H2O2-mediated oxidation Inhibition of tyrosinase in melanocyte extracts by

H2O2 To substantiate further the role of H2O2-mediated oxidation on Met 374 in the active site of the tyrosinase, To substantiate all above in vitro results further, we we used computer simulation. The active site of the determined tyrosinase activity in native HFM and EMC enzyme contains two copper atoms, CuA and CuB. extracts. HFMs yield a 1.6-fold higher activity compared Each is complexed by three histidines (180H, 202H, to EMCs (Fig. 5A). Our data reveal an almost complete 211H and 363H, 367H, 390H, respectively) to the ϫ ␣ absence of enzyme activity after H2O2-mediated oxida- protein matrix provided by 2 2 antiparallel -helices, tion of EMC extracts, supporting the in vitro results as ␣2.1, ␣2.2, ␣2.5, and ␣2.6, forming a 4-␣-helix bundle. shown with pure enzyme (Fig. 5B). The loop connecting the latter two ␣-helices contains

H2O2-MEDIATED OXIDATIVE STRESS IN GRAYING HAIR 2071 Figure 5. A) HFMs exhibit 1.5-fold higher tyrosinase activity compared to EMCs. Enzyme activity was determined in cell extracts from HFMs (Œ; nϭ2) and EMCs (ࡗ; nϭ2) following the l-dopachrome formation over time, as outlined in Materials and Methods. Rate of enzyme activity was calculated per milligram of protein per minute, yielding rates of 0.087 and 0.057 for HFMs ϫ Ϫ3 and EMCs, respectively. B) Tyrosinase activity is inactivated in EMC extracts in the presence of H2O2 (2 10 M). Enzyme activity was determined in cell extracts from EMCs following the l-dopachrome formation over time (ࡗ, native cell extract, nϭ2; f, oxidized cell extract, nϭ2) resulting in a 6.6-fold decrease after oxidation. Rate of enzyme activity was 0.066 vs. 0.010 mgϪ1 minϪ1 for native and oxidized EMC extract, respectively. the sequence 374MSQVQGS380 covering not only the distance to CuB did not change much. Thereafter, the active site. This condition is also important for enzyme distance between the partners of the original but function, as shown recently by mutations (29). The disrupted hydrogen bond between the HN␦1 of 367H peptide oxygen atoms of 377V and 374M serve as and the peptide oxygen of 374M fluctuated around 4 Å. hydrogen acceptors, and the HN␦1 groups of the imi- Compared to H367, all other histidines in the active site dazole rings of 180H and 367H serve as donors. In kept their original position and orientation very well. addition, another stabilizing hydrogen bond exists be- Consequently, enzymatic activity is impossible, since the tween the hydroxyl group of 380S and the peptide rotated 367H cannot orient the substrate in the appro- oxygen 374M. Thus, 367H and its backbone are stabi- priate way according to the proposed mechanism of lized with respect to position and orientation. The tyrosinase activity (50–52). 367H is important for the function, as it orients and In addition, the hydrogen bond between the peptide guides the substrate into the active site by a ␲-␲ oxygen of 377V and HN␦1 group of 180H increases to interaction between the imidazole ring and the phenyl 4.4 Å and will therefore break. This condition is a result ring of the substrate (50, 51). The proper orientation of of the above-mentioned connection of the oxygen of 367H is guaranteed by two hydrogen bonds, one fixing 374 Met-SϭO and the 367H, which pulls upwards the the backbone via an interaction between the peptide loop with 377V, while the methyl groups turn into the oxygen of 367H and the HO-group of 375S and the small cleft. Thus, 180H is affected, and therefore the CuA other connecting the HN␦1 group of 367H with the binding site would be destabilized as well (Fig. 6). peptide oxygen of 374M, which in turn builds up a hydrogen bond to 380S. H2O2-mediated oxidation shows no significant local DISCUSSION disturbance or sterical hindrance for the two Met-SϭO diastereomers. However, several MD simulations up to The in vivo identification of massive H2O2-concentra- a simulation time of 130 ps indicated that the structure tions in the gray hair shaft and the low catalase levels, as is most likely not stable. The oxygen from 374 Met-SϭO well as MSRA and MSRB activities in the entire hair is attracted by the 367H, independent of whether the R follicle, introduce a new step in the understanding of or S form of the Met-SϭO diastereomer was used in the human hair graying on the biochemical and molecular calculations. In most cases, the HN␦1 group of 367H biological level. It opens new windows for the preven- loses its original hydrogen bond to the peptide oxygen tion and possible reversal of this process. The com- of 374M within a short time, forming a new hydrogen pletely blunted Met-SϭO repair in the graying hair bond with the oxygen of 374 Met-SϭO that remains follicle offers several avenues for explanation of many very stable during the entire simulation time. The result structural altered proteins due to oxidation of Met is a major change in the orientation of the imidazole residues in the sequence. Clearly, this mechanism af- ring of 367H, which rotated up to 80°, while the contact fects tyrosinase, which in turn stops melanogenesis in

2072 Vol. 23 July 2009 The FASEB Journal WOOD ET AL. ϭ Figure 6. Homology modeling indicates severe alteration of H2O2-oxidized 374 Met-S O on the active site of hTyrosinase. The structure of the H2O2-oxidized enzyme is considerably altered compared to the native enzyme. The important amino acids are colored and indicated. Blue, copper atoms; red, oxygen atoms; black dashes, H-bonds. MD simulation is shown at 35 ps in comparison to the start.

HFMs. The scenario of the cascade is summarized in why and where these high levels of H2O2 are generated Fig. 7. However, the high levels of H2O2 present in during the hair graying process. Melanogenesis in the the hair shaft and hair follicle do not only oxidize hair follicle is certainly slowing down. Again, we use the free and bound Met residues. This ROS will also analogy with vitiligo. H2O2-mediated oxidation has affect bound and free Cys and Trp residues as well, as been documented for many other important regulators shown with oxidized tyrosinase by FT-Raman spec- of pigmentation, including the proopiomelanocortins troscopy (Fig. 4A). ␣-melanocyte-stimulating and ␤-endorphin While the entire hair follicle and the hair shaft are (53–55), the prohormone convertases (55), and the subject to H2O2-mediated stress, it is tempting to con- synthesis and recycling of the ubiquitous cofactor 6-tet- clude that this severely perturbed redox balance must rahydrobiopterin (56). These regulators are required precede melanocyte apoptosis and DNA damage, as in melanocytes for intracellular l-phenylalanine turn- documented earlier (4, 20). In this context it could be over to l-tyrosine (57), and many other pathways (for possible that, besides tyrosinase and MSRA and MSRB, review see ref. 11). It would be expected that the other proteins and peptides, including the antiapop- process of hair graying also affects these systems. Future totic Bcl-2 protein, are targets for oxidation, which in work needs to focus on these important mechanisms. turn could explain melanocyte apoptosis in the gray Although no data are currently available on the HFM hair follicle (4). Moreover, since low catalase levels and stem cell population, it is tempting to speculate that MSRA and MSRB are well explained by H2O2-mediated these cells may well also be target to oxidation. The oxidation (25, 26), the crucial open questions remain presence of MSRA and MSRB in all hair follicle cells

ϭ Figure 7. H2O2 prevents Met-S O repair in tyrosinase. In the brown hair follicle, H2O2 is generated in the micromolar range, which can activate transcription of many proteins, includ- ing catalase, tyrosinase, MSRA, and MSRB. Moreover, this ROS promotes enzyme activities in a dose-dependent manner (36, 58–60). In

the presence of millimolar H2O2 concentra- tions, oxidation of Met, Cys, Trp, and Sec residues in protein sequences are taking place, consequently altering the tertiary structures (26, 27). These structural changes often lead to deactivation of the affected protein/enzyme. This finding has been documented for catalase, MSRA, and MSRB (23, 37, 58, 61). Low catalase levels and activities have been documented in the gray hair follicle, which in turn leads to

increased H2O2 levels (43). Here, we provide evidence that tyrosinase activity is interrupted due to oxidation of Met 374 in the enzyme active site by this ROS. The resulting Met-SϭO cannot be repaired, because MSRA and MSRB are also deactivated by H2O2, as evidenced by low enzyme activities in gray hair follicle extracts, or they can originate from low protein levels. The same scenario applies for catalase. Taken together, a shift in the H2O2 redox balance can significantly alter melanogenesis in the human hair follicle.

H2O2-MEDIATED OXIDATIVE STRESS IN GRAYING HAIR 2073 certainly underlines the importance of Met-SϭO repair mediator Substance P–elucidating pathways involved in stress- in the entire hair follicle homeostasis. Most exciting is induced premature graying. Exp. Dermatol. 17, 632 10. Slominski, A., Tobin, D. J., Shibahara, S., and Wortsman, J. the observation that free l-methionine can prevent (2004) pigmentation in mammalian skin and its hor- Met-SϭO formation of protein- and peptide-bound Met monal regulation. Physiol. Rev. 84, 1155–1228 residues, saving in turn protein integrity and function, 11. Schallreuter, K. U., Bahadoran, P., Picardo, M., Slominski, A., Elassiuty, Y. E., Kemp, E. H., Giachino, C., Liu, J. B., Luiten, which could have great implications in the hair graying R. M., Lambe, T., Le Poole, I. C., Dammak, I., Onay, H., scenario in humans. Zmijewski, M. A., Dell’Anna, M. L., Zeegers, M. P., Cornall, R. J., Taken together, our data provide several concordant Paus, R., Ortonne, J. P., and Westerhof, W. (2008) Vitiligo lines of evidence, in vivo, in vitro, and in situ, in support pathogenesis: , genetic defect, excessive reactive oxygen species, calcium imbalance, or what else? Exp of a severely disturbed redox homeostasis/functionality Dermatol 17, 139–140; discussion 141–160 in all hair follicle cells, including the pigment-forming 12. Slominski, A., and Paus, R. (1993) Melanogenesis is coupled to melanocytes as well as in the hair shaft of the anagen murine anagen: toward new concepts for the role of melano- hair. Thus, concentration-dependent H O -mediated cytes and the regulation of melanogenesis in hair growth. 2 2 J. Invest. Dermatol. 101, 90S–97S oxidation of tyrosinase in HFMs, in association with the 13. Chase, H. B. (1954) Growth of the hair. Physiol. Rev. 34, 113–126 loss of functioning Met-SϭO repair, sheds a new light 14. Slominski, A., Wortsman, J., Plonka, P. M., Schallreuter, K. U., on the gradual slowing down of hair pigmentation as Paus, R., and Tobin, D. J. (2005) Hair follicle pigmentation. J. Invest. Dermatol. 124, 13–21 observed in the senile graying process. At this point, it 15. Nishimura, E. K., Granter, S. R., and Fisher, D. E. (2005) is tempting to focus on the blunted methionine repair Mechanisms of hair graying: incomplete melanocyte stem cell via MSRA and MSRB. Since the active site of MSRA has maintenance in the niche. Science 307, 720–724 two crucial Met residues, it would be interesting 16. Muller-Rover, S., Rossiter, H., Lindner, G., Peters, E. M., Kup- per, T. S., and Paus, R. (1999) Hair follicle apoptosis and Bcl-2. whether l-methionine could be useful. This theory is J. Investig. Dermatol. Symp. Proc. 4, 272–277 currently under investigation. A corrected repair offers 17. Veis, D. J., Sorenson, C. M., Shutter, J. R., and Korsmeyer, S. J. certainly an interesting target for the graying hair. (1993) Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75, 229–240 We dedicate this paper to the first author, who lost the 18. Nanninga, P. B., Ghanem, G. E., Lejeune, F. J., Bos, J. D., and battle against cancer during the preparation of this manu- Westerhof, W. (1991) Evidence for alpha-MSH binding sites on script in February 2008. A manuscript is a fitting final word for human scalp hair follicles: preliminary results. Pigment Cell Res. this renowned, enthusiastic scientist. This research has been 4, 193–198 supported by the University of Bradford, by a grant to K.U.S. 19. Slominski, A., Paus, R., Plonka, P., Chakraborty, A., Maurer, M., from Stiefel International, and by private donations. H.D. is Pruski, D., and Lukiewicz, S. 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