Functioning Methionine-S-Sulfoxide Reductases A and B Are ... - Core

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derma) and hair (poliosis), was known to the ancient Romans and is thought to be the first genetic disorder to have been recognized to exhibit autosomal.
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to the insight this model will provide for melanoma, it should also provide insight into the complex control of melanocytic migration and possibly the development of nevi. Kit remains a critically important regulatory molecule in melanocytic biology. CONFLICT OF INTEREST The author is a major shareholder in DigitalDerm, Inc. (total-body photography) and Malachite Corp. (medical databases).

REFERENCES Alexeev V, Yoon K (2006) Distinctive role of the cKit receptor tyrosine kinase signaling in mammalian melanocytes. J Investig Dermatol 126:1102–1110 Aoki H, Motohashi T, Yoshimura N, Yamazaki H, Yamane T, Panthier JJ et al. (2005) Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development. Dev Dyn 233:407–17 Blume-Jensen P, Siegbahn A, Stabel S, Heldin CH, Ronnstrand L (1993) Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C. EMBO J 12:4199–209 Grichnik JM, Burch JA, Burchette J, Shea CR (1998) The SCF/KIT pathway plays a critical role in the control of normal human melanocyte homeostasis. J Investig Dermatol 111:233–8 Ito M, Kawa Y, Ono H, Okura M, Baba T, Kubota Y et al. (1999) Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces apoptosis in murine melanocyte precursors. J Investig Dermatol 112:796–801 Grichnik JM, Burch JA, Singer S (2000) Inhibition of KIT in human nevus xenografts. J Investig Dermatol 114:788 (abstr.) Ito M, Kawa Y, Watabe H, Ono H, Ooka S, Nakamura M et al. (2004) Establishment by an original single-cell cloning method and characterization of an immortal mouse melanoblast cell line (NCCmelb4). Pigment Cell Res 17:643–50 Kunisada T, Lu SZ, Yoshida H, Nishikawa S, Nishikawa S, Mizoguchi M et al. (1998) Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor. J Exp Med 187:1565–73 Lin F, Burch JA, Singer S, Grichnik JM (2000) SCF induced proliferation of human melanocytes is masked in vitro by bFGF. J Investig Dermatol 114:859 (abstr.) Longley BJ, Reguera MJ, Ma Y (2001) Classes of c-KIT activating mutations: proposed mechanisms of action and implications for disease classification and therapy. Leuk Res 25:571–6 Ono H, Kawa Y, Asano M, Ito M, Takano A, Kubota Y et al. (1998) Development of melanocyte progenitors in murine Steel mutant neural crest explants cultured with stem cell factor, endothelin-3, or TPA. Pigment Cell Res 11:291–8 Pla P, Moore R, Morali OG, Grille S, Martinozzi

S, Delmas V et al. (2001) Cadherins in neural crest cell development and transformation. J Cell Physiol 189:121–32 Ronnstrand L (2004) Signal transduction via the stem cell factor receptor/c-Kit. Cell Mol Life Sci 61:2535–48 Sviderskaya EV, Wakeling WF, Bennett DC (1995) A cloned, immortal line of murine melanoblasts inducible to differentiate to melanocytes. Development 121:1547–57

Wehrle-Haller B, Weston JA (1995) Soluble and cell-bound forms of steel factor activity play distinct roles in melanocyte precursor dispersal and survival on the lateral neural crest migration pathway. Development 121:731–42 Yoshida H, Kunisada T, Kusakabe M, Nishikawa S, Nishikawa SI (1996) Distinct stages of melanocyte differentiation revealed by analysis of nonuniform pigmentation patterns. Development 122:1207–14

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Functioning Methionine-S-Sulfoxide Reductases A and B Are Present in Human Skin Karin U. Schallreuter1,2 Methionine residues in the structure of proteins and peptides are especially sensitive to oxidation by hydrogen peroxide (H2O2) yielding both the (R) and (S) diastereomers of methionine sulfoxide. This commentary shows that both diastereomers of methionine sulfoxide (R and S) can be repaired in the human epidermis by methionine sulfoxide reductases A and B, respectively. Journal of Investigative Dermatology (2006) 126, 947–949. doi:10.1038/sj.jid.5700086

Several lines of evidence provide support for the concept that oxidative stress causes aging and limits lifespan. Therefore, one crucial question arises: how does the human epidermis combat major oxidative stress–induced protein alterations? In this issue, Ogawa et al. (2006) describe the discovery of epidermal methionine-S-sulfoxide reductase A (MSRA), which contributes significantly to the presence of oxidative stress repair mechanisms in the human skin. Since hydrogen peroxide (H2O2) can oxidize the sulfur-containing methionine and cysteine residues in protein sequences, leading to disruption of protein structure and, in turn, to dysfunction, this commentary will put the specific problem of this reactive oxygen species (ROS)mediated damage to proteins and peptides into the context of the repair mechanisms involving MSRA and methionine-

S-sulfoxide reductase B (MSRB), both of which are essential to salvaging of protein structure and function as well as to cell viability. ROS oxidation of selective methionine, tryptophan, cysteine residues, and disulfide bridges in proteins and peptides

The epidermis is especially vulnerable to oxidative stress caused by multiple exogenous stimuli as well as by a plethora of endogenous metabolic events. Many mechanisms have been identified in controlling the maintenance of the redox balance in phase (Nordberg and Arner, 2001; Schallreuter and Wood, 2001). As is pointed out by Ogawa et al. (2006) and in earlier work from the same group, skin aging is also associated with a decreased capacity to neutralize ROS and to repair damaged proteins as well as DNA (Thiele et al., 1999). In this context Shigenaga

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Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, UK; 2Institute for Pigmentary Disorders in Association with EM Arndt University, Greifswald, Germany and University of Bradford, UK Correspondence: Prof. Karin U. Schallreuter, Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK. E-mail: [email protected]

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a

b

Figure 1. MSRA and MSRB are both expressed throughout the human epidermis. (a) In situ immunofluorescence of methionine sulfoxide reductases A and B shows partial co-localization of the two enzymes. Cryostat sections (5 µm) of human full skin biopsies were fixed in ice-cold methanol (6 min), rehydrated in phosphate-buffered saline (PBS) for 30 s, and blocked with normal donkey serum (NDS, 10% in PBS) for 90 min, then washed in PBS (5 min). Subsequently, sections were incubated overnight at 4 °C with the primary antibody (monoclonal anti-MSRB; mouse, 1:50 in 1% NDS; Autogen Bioclear UK Ltd., Calne, UK), washed 3× with PBS, incubated with FITC-labeled secondary antibody (donkey anti-mouse IgG, 1:100; Jackson ImmunoResearch Ltd., Soham, UK) for 60 min, washed 4× with PBS and once with PBS/Tween, blocked with NDS (10% in PBS) for 90 min, and then washed once in PBS for 5 min. MSRA was visualized using polyclonal anti-MSRA antibody (rabbit, 1:50 in 1% NDS; Autogen Bioclear UK Ltd, Calne, UK) for 3 h at RT, followed by a 3× wash in PBS, and was incubated with TRITC-labeled secondary antibody (donkey anti-rabbit IgG, 1:100; Jackson ImmunoResearch Ltd., Soham, UK) for 60 min, and then washed 4× with PBS and once with PBS/Tween. Slides were embedded in Vectashield Mounting Medium with 4’,6diamino-2-phenylindole (Vector, Peterborough, UK). Fluorescence pictures were taken with a Nikon Eclipse 80i microscope equipped with ACT-2U software (Nikon UK, Kingston, UK). (b) Western blotting confirmed the presence of MSRB (13 kDa calculated) in epidermal undifferentiated and differentiated keratinocyte cellular extracts. Proteins from primary epidermal undifferentiated and differentiated keratinocyte cell cultures were separated by SDS-PAGE (10%), transferred onto a polyvinylidene difluoride membrane, and incubated with the anti-MSRB antibody (1:2,000, overnight at 4 °C), followed by peroxidase-labeled antibody (goat anti-mouse IgG, 1:5,000, 1 h at RT; Sigma, Poole, UK). Bands were detected by ECL (Sigma, Poole, UK, and Eastman Kodak, Rochester, New York) at the expected size of 13 kDa.

and colleagues showed in 1994 that mitochondrial stability decreases with age owing to lipid peroxidation of mitochondrial membranes by ROS, which causes the release of cytochrome c from the electron transport chain, thus providing a signal for caspase-mediated apoptosis (Shigenaga et al., 1994). Very recently the role of ROS in the mitochon948

drion has received a great deal of attention in the context of aging in mammals, and it has been shown that overexpression of mitochondrial catalase prolongs lifespan in mice (Schriner et al., 2005). Moreover, reduced expression of MRSA decreased the lifespan by 40% in these animals, whereas its overexpression in Drosophila melanogaster leads to a 70%

Journal of Investigative Dermatology (2006), Volume 126

increase. Importantly, thioredoxin reductase/thioredoxin (TR/T) functions as the electron donor for both MSRA and MSRB (Kim and Gladyshev, 2004; Neiers et al., 2004), and overexpression of this system increased lifespan by 70% in the murine model (Mitsui et al., 2002). A great deal about oxidative stress in the human epidermis has been learned from studies of the depigmentation disorder vitiligo. The epidermis of individuals with vitiligo generates H2O2, in the concentration range of 10–3 M, continuously by various pathways leading to deactivation of catalase (EC 1.11.1.6), the enzyme that degrades this ROS to H2O and O2 (Schallreuter et al., 1999). More recently it has become clear that other important enzymes and proteins are also deactivated by this ROS as a result of oxidation of methionine residues to methionine sulfoxide in their structure. These proteins include dihydropteridine reductase (EC 1.6.99.7), an enzyme catalyzing the last step of (6R)L -erythro-5,6,7,8-tetrahydrobiopterin (6BH4) recycling, as well as acetylcholinesterase (EC 3.1.1.7). Deactivation also occurs through the oxidation of tryptophan residues, as observed in 4a-pterincarbinolamine dehydratase (EC 4.2.1.96), whereas oxidation of disulfide bridges by peracids greatly alters the structure of epidermal albumin (for review, see Schallreuter, 2005). Within the protein structure, methionine and cysteine are the residues most sensitive to oxidation by H2O2. This chemical oxidation yields roughly equal concentrations of the two diastereomers, (R) and (S), of methionine sulfoxide, resulting in the formation of intra- and intercellular disulfide bridges, respectively. MSRA and MSRB in the human epidermis

The purification and determination of Xray crystal structure of both MSRA and MSRB have been accomplished during the last 5 years and the catalytic mechanisms of action of the two proteins have been reported by several groups (Kim and Gladyshev, 2004; Neiers et al., 2004). MSRA specifically reduces methionine sulfoxide (S), whereas MSRB reduces methionine sulfoxide (R). Interestingly, the increased transcription and activity of MSRA by UVA light or by H2O2, as reported in this issue, mimics the transcriptional activation of TR (Nordberg

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and Arner, 2001). In this context it is fascinating that protein damage caused by H2O2-mediated oxidative stress is repaired by a system that is upregulated at the transcriptional level by this ROS. However, the presence of epidermal MSRA is not sufficient to recover oxidized methionines, due to the production of both the (R) and the (S) diastereomers of methionine sulfoxide by H2O2-mediated oxidation. It therefore was not surprising to find highly expressed and functional MSRB in the epidermal compartment in situ as well as in keratinocytes, as shown in Figure 1 (K. Rübsam, ongoing medical thesis, University of Hamburg). Hence, it

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Skin aging is also associated with a decreased capacity to neutralize ROS

can be concluded that proteins and peptides damaged as a result of the oxidation of methionines in their sequences can be totally salvaged by MSRA and MSRB. In this context it is noteworthy that both TR and the MSRs express cytosolic and mitochondrial membrane-integrated isoforms, thus maintaining mitochondrial as well as cytosolic stability (Hansel et al., 2002; Nordberg and Arner, 2001; Schallreuter and Wood, 2001).

presence and function of these reductases imply a functional TR/T system. Whether the reductases themselves may also be targets for oxidative stress remains to be determined. CONFLICT OF INTEREST The author states no conflict of interest.

REFERENCES Hansel A, Kuschel L, Hehl S, Lemke C, Agricola HJ, Hoshi T, Heinemann SH (2002) Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins. FASEB J 16:911–3 Kim HY, Gladyshev VN (2004) Methionine sulfoxide reduction in mammals: characterization of methionine-R-sulfoxide reductases. Mol Biol Cell 15:1055–64 Mitsui A, Hamuro J, Nakamura H, Kondo N, Hirabayashi Y, Ishizaki-Koizumi S et al. (2002) Overexpression of human thioredoxin in transgenic mice controls oxidative stress and life span. Antioxid Redox Signal 4:693–6 Neiers F, Kriznik A, Boschi-Muller S, Branlant G (2004) Evidence for a new sub-class of methionine sulfoxide reductases B with an alternative thioredoxin recognition signature. J Biol Chem 279:42462–8 Nordberg J, Arner ES (2001) Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med

31:1287–312 Ogawa F, Sander CS, Hansel A, Oehrl W, Kasperczyk, H, Elsner P et al. (2006) The repair enzyme peptide methionine-S-sulfoxide reductase is expressed in human epidermis and upregulated by UVA radiation. J Investig Dermatol 126:1128-34 Schallreuter KU, Moore J, Wood JM, Beazley WD, Gaze DC, Tobin DJ et al. (1999) In vivo and in vitro evidence for hydrogen peroxide (H2O2) accumulation in the epidermis of patients with vitiligo and its successful removal by a UVBactivated pseudocatalase. J Investig Dermatol Symp Proc 4:91–6 Schallreuter KU, Wood JM (2001) Thioredoxin reductase—its role in epidermal redox status. J Photochem Photobiol B 64:179–84 Schallreuter KU (2005) Vitiligo. In: Autoimmune Diseases of the Skin. Pathogenesis, Diagnosis, Management (Hertl, M. ed), Springer: Wien, 367–84 Schriner SE, Linford NJ, Martin GM, Treuting P, Ogburn CE, Emond M et al. (2005) Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 308:1909–11 Shigenaga MK, Hagen TM, Ames BN (1994) Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA 91:10771–8 Thiele JJ, Hsieh SN, Briviba K, Sies H (1999) Protein oxidation in human stratum corneum: susceptibility of keratins to oxidation in vitro and presence of a keratin oxidation gradient in vivo. J Investig Dermatol 113:335–9

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"Out, Damned Spot!" Richard A. Spritz1

The importance of epidermal TR/T for redox homeostasis

As early as in 1989 it was recognized that human epidermal keratinocytes express high levels of both cytosolic and membrane-integrated TR. This enzyme is subject to allosteric inhibition by calcium due to a single EF-hands binding site (for review see Schallreuter and Wood, 2001). Therefore, epidermal calcium homeostasis not only controls differentiation, but clearly also controls redox balance. The selenoenzyme TR, in addition to being the electron donor for both MSRs, this selenoenzyme has a very broad specificity, reducing disulfide bridges, H2O2, organoperoxides, vitamin K, and alloxan (Nordberg and Arner, 2001). In summary, the discovery of the MSRs in the skin adds yet another important role to the epidermal antioxidant repair machinery. Moreover, the

Mice transgenic for the Kit Val620Ala mutation, which in humans has been associated with progressive piebaldism, exhibit dominant white spotting but show no evidence of progressive depigmentation. These results are consistent with the previous hypothesis that progressive piebaldism might result from digenic inheritance, of the KITV620A mutation that causes piebaldism and a second, unknown locus that causes progressive depigmentation. Journal of Investigative Dermatology (2006) 126, 949–951. doi:10.1038/sj.jid.5700220

Lady Macbeth’s anguished lament speaks to some of the most puzzling conundrums in investigative dermatology. The white-spotting disorder piebaldism, because of its visually striking patches of white skin (leukoderma) and hair (poliosis), was known

to the ancient Romans and is thought to be the first genetic disorder to have been recognized to exhibit autosomal dominant inheritance (Morgan, 1786). We have learned a lot over the centuries. We now know that the leukodermal patches result from an almost

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Human Medical Genetics Program, University of Colorado Health Sciences Center, Aurora, Colorado, USA

Correspondence: Dr. Richard A. Spritz, Human Medical Genetics Program, University of Colorado Health Sciences Center, Mail-stop 8300, PO Box 6511, Aurora, Colorado 80045, USA. E-mail: [email protected]

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