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Cellular interactome dynamics during heat stress

ABSTRACT: Quantitative in vivo cross-linking-mass spectrometry was used to identify significant time-dependent interactome changes that occur prior to large-scale proteome abundance remodeling in cells subjected to heat stress. Interactome changes were identified within minutes of applied heat stress, including changes in chaperone systems as expected due to altered functional demand. Global analysis of all interactome changes revealed the largest significant enrichment in the class of RNA binding proteins, providing new time-dependent conformational insight on complex relationships that exist between transcription, translation and cellular stress response mechanisms upstream of transcriptional reprogramming. Observed interactome dynamics of RNA binding proteins that are also HSP90 clients constitute mechanistic changes that increase HSP90 functional demand, evident from observed HSP90 interactome changes early in heat stress. However, these changes together with observed heat stress-induced ATP level reduction result in observed complex time-dependent HSP90 interactome dynamics, indicating HSP90 conformational bottleneck formation with increased stress duration.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Sung-Gun Park

LAB HEAD: James E. Bruce

PROVIDER: PXD058191 | Pride | 2024-11-25

REPOSITORIES: Pride

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			Action	DRS
	20230911_10min_HeatShock_Frac_10_2nd_elution_rep1.raw	Raw
	20230911_10min_HeatShock_Frac_10_2nd_elution_rep2.raw	Raw
	20230911_10min_HeatShock_Frac_10_2nd_elution_rep3.raw	Raw
	20230911_10min_HeatShock_Frac_10_rep1.raw	Raw
	20230911_10min_HeatShock_Frac_10_rep2.raw	Raw

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Project description:Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative in vivo protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R2 values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type-specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.

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Cellular interactome dynamics during heat stress

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