yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis
Quality control-associated proteolysis (QCAP) is a fundamental mechanism that maintains cellular homeostasis by eliminating improperly folded proteins. In QCAP, the exposure of normally hidden <i>cis</i>-acting protein sequences, termed degrons, triggers misfolded protein ubiquitination,...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-06-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/13/6/987 |
| _version_ | 1797595844538531840 |
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| author | Bayan Mashahreh Shir Armony Tommer Ravid |
| author_facet | Bayan Mashahreh Shir Armony Tommer Ravid |
| author_sort | Bayan Mashahreh |
| collection | DOAJ |
| description | Quality control-associated proteolysis (QCAP) is a fundamental mechanism that maintains cellular homeostasis by eliminating improperly folded proteins. In QCAP, the exposure of normally hidden <i>cis</i>-acting protein sequences, termed degrons, triggers misfolded protein ubiquitination, resulting in their elimination by the proteasome. To identify the landscape of QCAP degrons and learn about their unique features we have developed an unbiased screening method in yeast, termed yGPS-P, which facilitates the determination of thousands of proteome-derived sequences that enhance proteolysis. Here we describe the fundamental features of the yGPS-P method and provide a detailed protocol for its use as a tool for degron search. This includes the cloning of a synthetic peptidome library in a fluorescence-based reporter system, and data acquisition, which entails the combination of Fluorescence-Activated Cell Sorting (FACS) and Next-Generation Sequencing (NGS). We also provide guidelines for data extraction and analysis and for the application of a machine-learning algorithm that established the evolutionarily conserved amino acid preferences and secondary structure propensities of QCAP degrons. |
| first_indexed | 2024-03-11T02:42:16Z |
| format | Article |
| id | doaj.art-e2bb61300ba34a54880a27e5f113f5ee |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-11T02:42:16Z |
| publishDate | 2023-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-e2bb61300ba34a54880a27e5f113f5ee2023-11-18T09:31:36ZengMDPI AGBiomolecules2218-273X2023-06-0113698710.3390/biom13060987yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated ProteolysisBayan Mashahreh0Shir Armony1Tommer Ravid2Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, IsraelDepartment of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, IsraelDepartment of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, IsraelQuality control-associated proteolysis (QCAP) is a fundamental mechanism that maintains cellular homeostasis by eliminating improperly folded proteins. In QCAP, the exposure of normally hidden <i>cis</i>-acting protein sequences, termed degrons, triggers misfolded protein ubiquitination, resulting in their elimination by the proteasome. To identify the landscape of QCAP degrons and learn about their unique features we have developed an unbiased screening method in yeast, termed yGPS-P, which facilitates the determination of thousands of proteome-derived sequences that enhance proteolysis. Here we describe the fundamental features of the yGPS-P method and provide a detailed protocol for its use as a tool for degron search. This includes the cloning of a synthetic peptidome library in a fluorescence-based reporter system, and data acquisition, which entails the combination of Fluorescence-Activated Cell Sorting (FACS) and Next-Generation Sequencing (NGS). We also provide guidelines for data extraction and analysis and for the application of a machine-learning algorithm that established the evolutionarily conserved amino acid preferences and secondary structure propensities of QCAP degrons.https://www.mdpi.com/2218-273X/13/6/987protein quality controlquality control-associated proteolysisubiquitin-proteasome system (UPS)degronomefluorescence-activated cell sorting (FACS)next generation sequencing (NGS) |
| spellingShingle | Bayan Mashahreh Shir Armony Tommer Ravid yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis Biomolecules protein quality control quality control-associated proteolysis ubiquitin-proteasome system (UPS) degronome fluorescence-activated cell sorting (FACS) next generation sequencing (NGS) |
| title | yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis |
| title_full | yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis |
| title_fullStr | yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis |
| title_full_unstemmed | yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis |
| title_short | yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis |
| title_sort | ygps p a yeast based peptidome screen for studying quality control associated proteolysis |
| topic | protein quality control quality control-associated proteolysis ubiquitin-proteasome system (UPS) degronome fluorescence-activated cell sorting (FACS) next generation sequencing (NGS) |
| url | https://www.mdpi.com/2218-273X/13/6/987 |
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