Spatially resolved profiling of protein conformation and interactions by biocompatible chemical cross-linking in living cells

ABSTRACT: The protein structures and interactions that maintain and regulate cellular processes in different subcellular organelles are heterogeneous and dynamic. However, it remains challenging to characterize the subcellular specificity and translocation of protein complexes in terms of conformation and interactions. Herein, we developed a spatially resolved protein complex profiling approach by biocompatible chemical cross-linking in living cells (SPACX) to monitor the dynamics of protein conformation, interactions and translocation. The advancement of fast capturing protein complexes in the physiological state, coupled with efficient enrichment of the cross-linked peptides, ensured deep-coverage analysis of the protein interactome in living cells. By ensemble structure refinement with cross-linking restraints, subcellular-specific conformation heterogeneity was identified for PTEN. PTEN displayed a broader range of dynamic conformation changes on the dual specificity domains in the nucleus than in the cytoplasm. Moreover, based on conformational differences, different interacting assemblies involving 128 cytoplasm-exclusively and 237 nucleus-exclusively PTEN-interacting proteins were found to account for diverse biological functions. Upon ubiquitin-proteasome system (UPS) stress, the assembly of PTEN and its interacting partners changed obviously during translocation. Both the components and interacting sites were dynamically identified in-vivo by SPACX approach coupled with subcellular isolation, and the corresponding functional pathways were highly enriched. Inspiringly, the distinguished interacting proteins among isoforms of PTEN and PTEN-L were accessible by the determination of sequence-unique cross-linking interfaces for direct interactions. All these results indicate the promise of SPACX to elucidate the functional heterogeneity of proteins in individual subcellular sociology.

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Cellular Organisms

SUBMITTER: Lihua Zhang 

PROVIDER: PXD043485 | JPOST Repository | Tue Jul 02 00:00:00 BST 2024

REPOSITORIES: jPOST