Conformation- specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution

• 成果类型: Article
• 署名作者: O'Leary, Kelly M.; Slezak, Tomasz; Kossiakoff, Anthony A.
• 署名单位: University of Chicago; University of Chicago
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14049
• DOI: 10.1073/pnas.2424679122
• 发表日期: 2025-06-17
• 关键词: growth; inhibition; rna; longevity; mechanism; PATHWAY; complex; raptor

摘要

Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12-Rapamycin binding site of mTOR (mTORFRB). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTORFRB through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTORFRB. Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTORFRB. Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.