Buckwheat Trypsin Inhibitor Prolongs the Healthy Lifespan by Activating Mitochondrial Protein Import Stress in Caenorhabditis elegans

Mitochondria are well-established as pivotal regulators of organismal aging, yet the mechanistic connection between mitochondrial protein translocation and the aging remained elusive. Previous studies have shown that recombinant buckwheat trypsin inhibitor (rBTI) extends the lifespan of Caenorhabditis elegans and enhances stress resistance, and structural characterization reveals an α-helix (residues 18-28) within rBTI that shares notable sequence similarity to presequence of mitochondrial aldehyde dehydrogenase (pALDH). Building on these observations, the present study identifies rBTI as a novel mitochondrial-targeting protein induces mitochondrial protein import stress. Functional validation through site-directed mutagenesis confirms that two positively charged residues (K20 and K23) within this α-helix are indispensable for lifespan extension and ATP production enhancement. Mechanistic investigations further demonstrate that mitophagy contributes to rBTI-mediated lifespan extension, while the mitochondrial chaperone protein Hsp 60 is not required for this process. Using genetic loss-of-function experiments, we further identify TOMM-20 and TOMM-22, core components of the outer mitochondrial membrane translocase complex, as direct target of rBTI. Ablation of either TOMM-20 or TOMM-22 completely abolishes the lifespan-extending effects of rBTI, confirming their essential role in mediating rBTI's biological activity. Metabolomic profiling further reveals that rBTI triggered the mitochondrial protein import stress triggers systemic metabolic reprogramming, with rewiring of amino acid and lipid metabolism pathways as key drivers of enhanced longevity. Taken together, our findings elucidate a novel molecular mechanism by which rBTI promotes healthy aging, and highlight the critical role of mitochondrial protein import homeostasis in aging regulation. These results also underscore the potential of targeting mitochondrial protein import pathways for the development of innovative anti-aging interventions.