Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii
Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the pathways and molecular mechanisms perturbed by such compounds requires t...
| Main Authors: | , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Bio-Protocol, LLC
2023
|
| Online Access: | https://hdl.handle.net/1721.1/147078 |
| _version_ | 1826190283068407808 |
|---|---|
| author | Herneisen, Alice Lourido, Sebastian |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Herneisen, Alice Lourido, Sebastian |
| author_sort | Herneisen, Alice |
| collection | MIT |
| description | Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the pathways and molecular mechanisms perturbed by such compounds requires target deconvolution. In parasites, such deconvolution has been achieved via chemogenomic approaches-for example, directed evolution followed by whole-genome sequencing or genome-wide knockout screens. As a proteomic alternative that directly probes the physical interaction between compound and protein, thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), recently emerged as a method to identify small molecule-target interactions in living cells and cell extracts in a variety of organisms, including unicellular eukaryotic pathogens. Ligand binding induces a thermal stability shift-stabilizing or destabilizing proteins that change conformationally in response to the ligand-that can be measured by mass spectrometry (MS). Cells are incubated with different concentrations of ligand and heated, causing thermal denaturation of proteins. The soluble protein is extracted and quantified with multiplexed, quantitative MS, resulting in thousands of thermal denaturation profiles. Proteins engaging the ligand can be identified by their compound-dependent thermal shift. The protocol provided here can be used to identify ligand-target interactions and assess the impact of environmental or genetic perturbations on the thermal stability of the proteome in T. gondii and other eukaryotic pathogens. Graphic abstract: Thermal proteome profiling for target identification in the apicomplexan parasite T. gondii. |
| first_indexed | 2024-09-23T08:37:53Z |
| format | Article |
| id | mit-1721.1/147078 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T08:37:53Z |
| publishDate | 2023 |
| publisher | Bio-Protocol, LLC |
| record_format | dspace |
| spelling | mit-1721.1/1470782023-01-13T03:48:04Z Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii Herneisen, Alice Lourido, Sebastian Massachusetts Institute of Technology. Department of Biology Toxoplasma gondii is a single-celled eukaryotic parasite that chronically infects a quarter of the global population. In recent years, phenotypic screens have identified compounds that block parasite replication. Unraveling the pathways and molecular mechanisms perturbed by such compounds requires target deconvolution. In parasites, such deconvolution has been achieved via chemogenomic approaches-for example, directed evolution followed by whole-genome sequencing or genome-wide knockout screens. As a proteomic alternative that directly probes the physical interaction between compound and protein, thermal proteome profiling (TPP), also known as the cellular thermal shift assay (CETSA), recently emerged as a method to identify small molecule-target interactions in living cells and cell extracts in a variety of organisms, including unicellular eukaryotic pathogens. Ligand binding induces a thermal stability shift-stabilizing or destabilizing proteins that change conformationally in response to the ligand-that can be measured by mass spectrometry (MS). Cells are incubated with different concentrations of ligand and heated, causing thermal denaturation of proteins. The soluble protein is extracted and quantified with multiplexed, quantitative MS, resulting in thousands of thermal denaturation profiles. Proteins engaging the ligand can be identified by their compound-dependent thermal shift. The protocol provided here can be used to identify ligand-target interactions and assess the impact of environmental or genetic perturbations on the thermal stability of the proteome in T. gondii and other eukaryotic pathogens. Graphic abstract: Thermal proteome profiling for target identification in the apicomplexan parasite T. gondii. 2023-01-12T14:41:20Z 2023-01-12T14:41:20Z 2021 2023-01-12T14:36:54Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/147078 Herneisen, Alice and Lourido, Sebastian. 2021. "Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii." BIO-PROTOCOL, 11 (21). en 10.21769/BIOPROTOC.4207 BIO-PROTOCOL Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Bio-Protocol, LLC Prof. Lourido |
| spellingShingle | Herneisen, Alice Lourido, Sebastian Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii |
| title | Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii |
| title_full | Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii |
| title_fullStr | Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii |
| title_full_unstemmed | Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii |
| title_short | Thermal Proteome Profiling to Identify Protein-ligand Interactions in the Apicomplexan Parasite Toxoplasma gondii |
| title_sort | thermal proteome profiling to identify protein ligand interactions in the apicomplexan parasite toxoplasma gondii |
| url | https://hdl.handle.net/1721.1/147078 |
| work_keys_str_mv | AT herneisenalice thermalproteomeprofilingtoidentifyproteinligandinteractionsintheapicomplexanparasitetoxoplasmagondii AT louridosebastian thermalproteomeprofilingtoidentifyproteinligandinteractionsintheapicomplexanparasitetoxoplasmagondii |