Abstract
Mitochondrial stress activates the integrated stress response (ISR) through the mitochondrial protein DELE1, which relays stress signals to the cytosolic kinase HRI to induce ATF4. Dysregulation of DELE1-mediated signaling has been implicated in pathological conditions, yet molecular strategies to modulate DELE1 remain unavailable. Here, we report de novo designed proteins that bind DELE1, block its oligomerization, and inhibit DELE1-mediated ISR activation. Several designs form stable complexes with DELE1 and disrupt its oligomerization in vitro while preserving DELE1's ability to bind HRI. In cells, these designs suppress ATF4 induction during mitochondrial stress and impair the recovery of elongated mitochondrial morphology following transient insult. Crystal structure analysis, structural modeling, and targeted mutagenesis confirm that the designed proteins engage a critical interface required for DELE1 oligomerization. These findings establish DELE1 as a druggable target and demonstrate that de novo designed proteins offer precise tools to modulate this pathway, laying groundwork for therapeutic development.