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Integrated Proteogenomic Approach for Identifying Degradation Motifs in Eukaryotic Cells

Part of the Methods in Molecular Biology book series (MIMB,volume 1844)

Abstract

Since its discovery nearly 40 years ago, many components of the ubiquitin-proteasome system (UPS) have been identified and characterized in detail. However, a key aspect of the UPS that remains largely obscure is the signals that initiate the interaction of a substrate with enzymes of the UPS machinery. Understanding these signals is of particular interest for studies that examine the mechanism of substrate recognition for proteins that have adopted a non-native structure, as part of the cellular protein quality control (PQC) defense mechanism. Such studies are quite salient as the entire proteome makes up the potential battery of PQC substrates, and yet only a limited number of ubiquitination pathways are known to handle misfolded proteins. Our current research aims at understanding how a small number of PQC ubiquitin-protein ligases specifically recognize and ubiquitinate the overwhelming assortment of misfolded proteins. Here, we present a new proteogenomic approach for identifying and characterizing recognition motifs within degradation elements (degrons) in a high-throughput manner. The method utilizes yeast growth under restrictive conditions for selecting protein fragments that confer instability. The corresponding cDNA fragments are analyzed by next-generation sequencing (NGS) that provides information about each fragment’s identity, reading frame, and abundance over time. This method was used by us to identify PQC-specific and compartment-specific degrons. It can readily be modified to study protein degradation signals and pathways in other organisms and in various settings, such as different strain backgrounds and under various cell conditions, all of which can be sequenced and analyzed simultaneously.

Key words

  • Ubiquitin-proteasome system
  • Protein quality control
  • Degradation signals
  • Proteogenomics
  • Next-generation sequencing
  • Yeast competition assay

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Correspondence to Richard G. Gardner or Tommer Ravid .

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Geffen, Y., Appleboim, A., Gardner, R.G., Ravid, T. (2018). Integrated Proteogenomic Approach for Identifying Degradation Motifs in Eukaryotic Cells. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_9

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  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8705-4

  • Online ISBN: 978-1-4939-8706-1

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