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Reactive Oxygen Species (ROS) Protection via Cysteine Oxidation in the Epidermal Cornified Cell Envelope

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Book cover Epidermal Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1195))

Abstract

The outermost layer of our skin functions as a barrier to protect us from physical, chemical, and biological environmental insults. This protective function is mediated by the epidermal cornified cell envelope (CE) which serves both as a mechanical and permeability barrier. Recently we have discovered that the CE constitutes also a first-line antioxidant shield which relies greatly on cysteine residues in CE precursor proteins. Here we describe methods and protocols to study the cysteine-mediated antioxidant function of the CE at the level of the whole organ (the skin), individual cells (keratinocytes), or isolated proteins (SPRR family).

An erratum to this chapter can be found at http://dx.doi.org/10.1007/7651_2013_66

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References

  1. Dickinson BC, Chang CJ (2011) Chemistry and biology of reactive oxygen species in signaling or stress responses. Nat Chem Biol 7(8):504–511. doi:nchembio.607 [pii] 10.1038/nchembio.607

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Hoeijmakers JH (2009) DNA damage, aging, and cancer. N Engl J Med 361(15):1475–1485. doi:361/15/1475 [pii] 10.1056/NEJMra0804615

    Article  CAS  PubMed  Google Scholar 

  3. Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G (2013) The hallmarks of aging. Cell 153(6):1194–1217. doi:S0092-8674(13)00645-4 [pii] 10.1016/j.cell.2013.05.039

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Harman D (1956) Aging: a theory based on free radical and radiation chemistry. J Gerontol 11(3):298–300

    Google Scholar 

  5. Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408(6809):239–247. doi:10.1038/35041687

    Article  CAS  PubMed  Google Scholar 

  6. Jacob KD, Noren Hooten N, Tadokoro T, Lohani A, Barnes J, Evans MK (2013) Alzheimer’s disease-associated polymorphisms in human OGG1 alter catalytic activity and sensitize cells to DNA damage. Free Radic Biol Med 63:115–125. doi:S0891-5849(13)00220-7 [pii] 10.1016/j.freeradbiomed.2013.05.010

    Article  CAS  PubMed  Google Scholar 

  7. Paget MS, Buttner MJ (2003) Thiol-based regulatory switches. Annu Rev Genet 37:91–121. doi:10.1146/annurev.genet.37.110801.142538

    Article  CAS  PubMed  Google Scholar 

  8. Winterbourn CC, Hampton MB (2008) Thiol chemistry and specificity in redox signaling. Free Radic Biol Med 45(5):549–561. doi:S0891-5849(08)00280-3 [pii] 10.1016/j.freeradbiomed.2008.05.004

    Article  CAS  PubMed  Google Scholar 

  9. Winyard PG, Moody CJ, Jacob C (2005) Oxidative activation of antioxidant defence. Trends Biochem Sci 30(8):453–461. doi:S0968-0004(05)00180-5 [pii] 10.1016/j.tibs.2005.06.001

    Article  CAS  PubMed  Google Scholar 

  10. D'Autreaux B, Toledano MB (2007) ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat Rev Mol Cell Biol 8(10):813–824. doi:nrm2256 [pii] 10.1038/nrm2256

    Article  PubMed  Google Scholar 

  11. Vermeij WP, Backendorf C (2010) Skin cornification proteins provide global link between ROS detoxification and cell migration during wound healing. PLoS One 5(8):e11957. doi:10.1371/journal.pone.0011957

    Article  PubMed Central  PubMed  Google Scholar 

  12. Vermeij WP, Alia A, Backendorf C (2011) ROS quenching potential of the epidermal cornified cell envelope. J Invest Dermatol 131(7):1435–1441. doi:jid2010433 [pii] 10.1038/jid.2010.433

    Article  CAS  PubMed  Google Scholar 

  13. Candi E, Schmidt R, Melino G (2005) The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 6(4):328–340. doi:nrm1619 [pii] 10.1038/nrm1619

    Article  CAS  PubMed  Google Scholar 

  14. Kalinin AE, Kajava AV, Steinert PM (2002) Epithelial barrier function: assembly and structural features of the cornified cell envelope. Bioessays 24(9):789–800. doi:10.1002/bies.10144

    Article  CAS  PubMed  Google Scholar 

  15. Schafer M, Farwanah H, Willrodt AH, Huebner AJ, Sandhoff K, Roop D, Hohl D, Bloch W, Werner S (2012) Nrf2 links epidermal barrier function with antioxidant defense. EMBO Mol Med 4(5):364–379. doi:10.1002/emmm.201200219

    Article  PubMed Central  PubMed  Google Scholar 

  16. Huebner AJ, Dai D, Morasso M, Schmidt EE, Schafer M, Werner S, Roop DR (2012) Amniotic fluid activates the nrf2/keap1 pathway to repair an epidermal barrier defect in utero. Dev Cell 23(6):1238–1246. doi:S1534-5807(12)00522-9 [pii] 10.1016/j.devcel.2012.11.002

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Cabral A, Voskamp P, Cleton-Jansen AM, South A, Nizetic D, Backendorf C (2001) Structural organization and regulation of the small proline-rich family of cornified envelope precursors suggest a role in adaptive barrier function. J Biol Chem 276(22):19231–19237. doi:10.1074/jbc.M100336200 M100336200 [pii]

    Article  CAS  PubMed  Google Scholar 

  18. Vermeij WP, Florea BI, Isenia S, Alia A, Brouwer J, Backendorf C (2012) Proteomic Identification of in Vivo Interactors Reveals Novel Function of Skin Cornification Proteins. J Proteome Res. doi:10.1021/pr300310b

    PubMed  Google Scholar 

  19. Dickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, Louis DN, Li FP, Rheinwald JG (2000) Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 20(4):1436–1447

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Mastroberardino PG, Orr AL, Hu X, Na HM, Greenamyre JT (2008) A FRET-based method to study protein thiol oxidation in histological preparations. Free Radic Biol Med 45(7):971–981. doi:S0891-5849(08)00364-X [pii] 10.1016/j.freeradbiomed.2008.06.018

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. Kalb VF Jr, Bernlohr RW (1977) A new spectrophotometric assay for protein in cell extracts. Anal Biochem 82(2):362–371

    Google Scholar 

  22. Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856–2860. doi:nprot.2006.468 [pii] 10.1038/nprot.2006.468

    Article  CAS  PubMed  Google Scholar 

  23. Florea BI, Verdoes M, Li N, van der Linden WA, Geurink PP, van den Elst H, Hofmann T, de Ru A, van Veelen PA, Tanaka K, Sasaki K, Murata S, den Dulk H, Brouwer J, Ossendorp FA, Kisselev AF, Overkleeft HS (2010) Activity-based profiling reveals reactivity of the murine thymoproteasome-specific subunit beta5t. Chem Biol 17(8):795–801. doi:S1074-5521(10)00257-7 [pii] 10.1016/j.chembiol.2010.05.027

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Mehrel T, Hohl D, Rothnagel JA, Longley MA, Bundman D, Cheng C, Lichti U, Bisher ME, Steven AC, Steinert PM et al (1990) Identification of a major keratinocyte cell envelope protein, loricrin. Cell 61(6):1103–1112. doi:0092-8674(90)90073-N [pii]

    Article  CAS  PubMed  Google Scholar 

  25. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906. doi:nprot.2007.261 [pii] 10.1038/nprot.2007.261

    Article  CAS  PubMed  Google Scholar 

  26. Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20(18):3551–3567. doi:10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2–2 [pii] 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2–2

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank all present and former members of the Molecular Cell Signaling group at Molecular Genetics (Leiden Institute of Chemistry), who have contributed to the establishment and testing of the protocols described here, and all current members of the Aging group at Genetics (ErasmusMC, Rotterdam). Bobby Florea (Biosyn, LIC) is acknowledged for his help with the mass spectrometer.

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Authors and Affiliations

  1. Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, NL- 2333CC, Leiden, The Netherlands

    Claude Backendorf

  2. University of Leiden, Leiden, The Netherlands

    Wilbert P. Vermeij

Authors
  1. Wilbert P. Vermeij
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  2. Claude Backendorf
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Corresponding author

Correspondence to Claude Backendorf .

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Editors and Affiliations

  1. Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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Vermeij, W.P., Backendorf, C. (2013). Reactive Oxygen Species (ROS) Protection via Cysteine Oxidation in the Epidermal Cornified Cell Envelope. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 1195. Springer, New York, NY. https://doi.org/10.1007/7651_2013_51

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  • DOI: https://doi.org/10.1007/7651_2013_51

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-1223-0

  • Online ISBN: 978-1-4939-1224-7

  • eBook Packages: Springer Protocols

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