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High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis
Published on: October 15, 2019
Proteomic analysis of complexes formed by human topoisomerase I
Alicja Czubaty1, Agnieszka Girstun, Barbara Kowalska-Loth
1Institute of Biochemistry, Warsaw University, ul. Miecznikowa 1, 02-096 Warszawa, Poland.
Insights
Researchers identified 36 nuclear proteins interacting with human topoisomerase I, primarily involved in RNA metabolism. The N-terminal and cap regions of topoisomerase I are key binding sites, with RRM domains crucial for interaction.
Area of Science:
- Molecular Biology
- Proteomics
- Biochemistry
Background:
- Human topoisomerase I is a nuclear enzyme crucial for DNA relaxation and SR protein phosphorylation.
- It engages in various protein-protein interactions, suggesting a broader functional role.
- Understanding these interactions is key to elucidating topoisomerase I's cellular functions.
Purpose of the Study:
- To perform a comprehensive proteomic analysis to identify protein partners of human topoisomerase I.
- To map the specific regions of topoisomerase I involved in these protein interactions.
- To characterize the functional roles of the identified interacting proteins.
Main Methods:
- Utilized co-immunoprecipitation with anti-topoisomerase I antibodies on HeLa nuclear extracts.
- Employed affinity chromatography with topoisomerase I fragments and mass spectrometry for protein identification.
- Performed docking calculations to analyze protein-domain interactions.
Main Results:
- Identified 36 nuclear proteins associated with topoisomerase I, including 29 novel partners.
- The N-terminal domain and the cap region of the core domain were identified as primary binding sites.
- A significant portion of cap-binding proteins possess RRM domains, implicated in interaction with the cap region.
Conclusions:
- Human topoisomerase I interacts with a diverse set of nuclear proteins, predominantly involved in RNA metabolism.
- The N-terminal and cap regions are critical interaction hubs for topoisomerase I.
- RRM domains are likely key mediators of interaction between topoisomerase I and associated proteins.
Abstract:
Human topoisomerase I is a nuclear enzyme that catalyses DNA relaxation and phosphorylation of SR proteins. Topoisomerase I participates in several protein-protein interactions. We performed a proteomic analysis of protein partners of topoisomerase I. Two methods were applied to proteins of the nuclear extract of HeLa cells: a co-immunoprecipitation and an affinity chromatography combined with mass spectrometry. Complexes formed by topoisomerase I with its protein partners were immunoprecipitated by scleroderma anti-topoisomerase I antibodies. To identify binding sites for the protein partners, baits corresponding to fragments of topoisomerase I were constructed and used in the affinity chromatography. The N-terminal domain and the cap region of the core domain appeared to be the main regions that bound proteins. We identified 36 nuclear proteins that were associated with topoisomerase I. The proteins were mainly involved in RNA metabolism. We found 29 new and confirmed 7 previously identified protein partners of topoisomerase I. More than 40% proteins that associate with the cap region contain two closely spaced RRM domains. Docking calculations identified the RRM domains as a possible site for the interaction of these proteins with the cap region.
