Background: Intracellular proteolysis is a fundamental regulatory mechanism. Lon, a highly conserved ATP-dependent AAA+ protease, is a central hub for protein quality control and stress survival. While critical for bacterial pathogenicity and human mitochondrial homeostasis, the precise mechanisms governing its regulation remain elusive. Our laboratory has established robust proteomics approaches to identify Lon substrates (Omnus & Fink et al. 2021) and characterized novel allosteric regulators, LarA and HspQ (Omnus & Fink et al. 2023, Omnus et al., in preparation), which modulate substrate selectivity via NTD-binding. After focusing for the past 13 years on bacterial Lon, we recently received funding from Cancerfonden to extend our work to human mitochondrial Lon (LONP1)—a protease linked to cancer, aging, and neurodegenerative diseases. Although the Lon NTD is structurally conserved, regulators for mitochondrial Lon remain largely unknown. In the first part of our NAISS project, we conducted proteome-wide AF3 screenings for interactors of LONP1 and Lon protease in yeast (Pim1). This screen resulted in a group of high confidence interactors. Notably, among these hits were several proteins that were recently demonstrated to interact with Lon in vivo (Bertgen et al. 2024), demonstrating that our AF3-based screen was successful in retrieving true Lon interactors. Project Goal: This project will expand our discovery pipeline by integrating de novo peptide binder design as a parallel strategy to interrogate Lon regulation. Alongside the wet-lab validation of AF3-identified interactors, we will leverage BindCraft, a state-of-the-art generative deep-learning framework (Pacesa et al. 2025), to engineer synthetic peptide binders targeting the Lon NTD for three evolutionary tiers (Caulobacter, yeast, and human). This dual approach—validating natural partners while designing synthetic modulators—could offer unique insights into how the NTD regulates the functional landscape and substrate binding of the Lon hexamer. Our Approach: We plan to employ a "function-first" pipeline: In Silico Design and Orthogonal Validation: Utilizing BindCraft to generate peptides targeting the conserved NTD regulatory pocket. While BindCraft includes rigorous internal filtering (ProteinMPNN/Rosetta/AF2), we will implement an additional AF3-based validation layer to re-rank candidates and ensure structural complementarity. Functional Screening: Rather than screening for binding affinity, we will directly test candidates for biological function. We will measure the modulation of Lon activity and substrate degradation rates biochemically in the presence of designed binders by using enzymatic assays. Mechanistic Validation: We expect to demonstrate that NTD-specific binding modulates the conformational state of the Lon hexamer, thereby altering substrate recognition and turnover rates. Project Impact: This work aims to advance our research from observing interactions to the rational modulation of proteolytic machines. Scientific Insight: Using natural and designed probes to decouple communication within the Lon hexamer will reveal how the NTD controls substrate selection across species. Therapeutic Potential: Human LONP1 designs could serve as scaffolds for modulating Lon in cancer and age-related diseases. Innovation: Combining AF3-driven discovery with BindCraft-driven design establishes a model for using state-of-the-art tools to dissect and modulate complex systems.