Identification of covalent modifications regulating immune signaling complex composition and phenotype
Abstract Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post‐transcriptional modifications regulating signaling complex composition and functional phenotypes in one...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Springer Nature
2021-07-01
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| Series: | Molecular Systems Biology |
| Subjects: | |
| Online Access: | https://doi.org/10.15252/msb.202010125 |
| _version_ | 1797284632058658816 |
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| author | Annika Frauenstein Stefan Ebner Fynn M Hansen Ankit Sinha Kshiti Phulphagar Kirby Swatek Daniel Hornburg Matthias Mann Felix Meissner |
| author_facet | Annika Frauenstein Stefan Ebner Fynn M Hansen Ankit Sinha Kshiti Phulphagar Kirby Swatek Daniel Hornburg Matthias Mann Felix Meissner |
| author_sort | Annika Frauenstein |
| collection | DOAJ |
| description | Abstract Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post‐transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP‐APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS‐based quantification. We report the time‐resolved interplay of more than 50 previously undescribed modification and hundreds of protein–protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site‐specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll‐like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost‐effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells. |
| first_indexed | 2024-03-07T17:50:49Z |
| format | Article |
| id | doaj.art-59400247e5b546bb933065d3ad982aa7 |
| institution | Directory Open Access Journal |
| issn | 1744-4292 |
| language | English |
| last_indexed | 2024-03-07T17:50:49Z |
| publishDate | 2021-07-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj.art-59400247e5b546bb933065d3ad982aa72024-03-02T14:01:50ZengSpringer NatureMolecular Systems Biology1744-42922021-07-01177n/an/a10.15252/msb.202010125Identification of covalent modifications regulating immune signaling complex composition and phenotypeAnnika Frauenstein0Stefan Ebner1Fynn M Hansen2Ankit Sinha3Kshiti Phulphagar4Kirby Swatek5Daniel Hornburg6Matthias Mann7Felix Meissner8Experimental Systems Immunology Max Planck Institute of Biochemistry Martinsried GermanyExperimental Systems Immunology Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyExperimental Systems Immunology Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Molecular Machines and Signaling Max Planck Institute of Biochemistry Martinsried GermanyDepartment of Genetics School of Medicine Stanford University Stanford CA USADepartment of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried GermanyExperimental Systems Immunology Max Planck Institute of Biochemistry Martinsried GermanyAbstract Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post‐transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP‐APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS‐based quantification. We report the time‐resolved interplay of more than 50 previously undescribed modification and hundreds of protein–protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site‐specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll‐like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost‐effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells.https://doi.org/10.15252/msb.202010125mass spectrometryposttranslational modificationsprotein interactionsproteomicssignaling networks |
| spellingShingle | Annika Frauenstein Stefan Ebner Fynn M Hansen Ankit Sinha Kshiti Phulphagar Kirby Swatek Daniel Hornburg Matthias Mann Felix Meissner Identification of covalent modifications regulating immune signaling complex composition and phenotype Molecular Systems Biology mass spectrometry posttranslational modifications protein interactions proteomics signaling networks |
| title | Identification of covalent modifications regulating immune signaling complex composition and phenotype |
| title_full | Identification of covalent modifications regulating immune signaling complex composition and phenotype |
| title_fullStr | Identification of covalent modifications regulating immune signaling complex composition and phenotype |
| title_full_unstemmed | Identification of covalent modifications regulating immune signaling complex composition and phenotype |
| title_short | Identification of covalent modifications regulating immune signaling complex composition and phenotype |
| title_sort | identification of covalent modifications regulating immune signaling complex composition and phenotype |
| topic | mass spectrometry posttranslational modifications protein interactions proteomics signaling networks |
| url | https://doi.org/10.15252/msb.202010125 |
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