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Effects of the Extracellular Matrix on the Proteome of Primary Skin Fibroblasts

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Skin Tissue Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1993))

Abstract

The cellular microenvironment often plays a crucial role in disease development and progression. In recessive dystrophic epidermolysis bullosa (RDEB), biallelic mutations of the gene COL7A1, encoding for collagen VII, the main component of anchoring fibrils, lead to a loss of collagen VII in the extracellular matrix (ECM). Loss of collagen VII in skin is linked to a destabilization of the dermal-epidermal junction zone, blister formation, chronic wounds, fibrosis, and aggressive skin cancer. Thus, RDEB cells can serve as a model system to study the effects of a perturbed ECM on the cellular proteome. In this chapter, we describe in detail the combination of stable isotope labeling by amino acids in cell culture (SILAC) of primary skin fibroblasts with reseeding of fibroblasts on decellularized collagen VII-positive and -negative ECM to study the consequences of collagen VII loss on the cellular proteome. This approach allows the quantitative, time-resolved analysis of cellular protein dynamics in response to ECM perturbation by liquid chromatography-mass spectrometry.

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Acknowledgments

We thank all members of the Protein Homeostasis laboratory of Prof. Jörn Dengjel for their help and discussions. We thank Prof. Leena Bruckner-Tuderman and the Molecular Dermatology laboratory of the University Clinic Freiburg i. Br. for providing the fibroblasts. This research was supported by the German Research Foundation (DFG) through grant DE 1757/3-2 and by the Swiss National Science Foundation (SNSF) through grant 31003A-166482/1.

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Authors and Affiliations

  1. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Regine C. Tölle & Jörn Dengjel

  2. Department of Dermatology, Medical Center – University of Freiburg, Freiburg, Germany

    Regine C. Tölle & Jörn Dengjel

Authors
  1. Regine C. Tölle
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  2. Jörn Dengjel
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Correspondence to Jörn Dengjel .

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Editors and Affiliations

  1. University Children’s Hospital Zurich, Zurich, Switzerland

    Sophie Böttcher-Haberzeth  & Thomas Biedermann  & 

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Tölle, R.C., Dengjel, J. (2019). Effects of the Extracellular Matrix on the Proteome of Primary Skin Fibroblasts. In: Böttcher-Haberzeth, S., Biedermann, T. (eds) Skin Tissue Engineering. Methods in Molecular Biology, vol 1993. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9473-1_15

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  • DOI: https://doi.org/10.1007/978-1-4939-9473-1_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9472-4

  • Online ISBN: 978-1-4939-9473-1

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