Title:	An AlphaFold3-driven in silico screen to identify novel protein-protein interacting partners of the human LONP1 protease, and experimental validation of screening results in a wet lab setting.
DNr:	NAISS 2025/22-1088
Project Type:	NAISS Small Compute
Principal Investigator:	Efstathios Nikolaos Vlachos <nikolaos.vlachos-efstathios@su.se>
Affiliation:	Stockholms universitet
Duration:	2025-08-19 – 2026-03-01
Classification:	10616
Homepage:	https://jonaslab.org/
Keywords:

Abstract

Background: Intracellular proteolysis represents an efficient way to regulate the levels of specific proteins. Lon is a highly conserved ATP-dependent protease that has important regulatory and protein quality control functions in cells from the three domains of life. In bacteria, Lon is critical for stress survival, antibiotic tolerance and pathogenicity, while in eukaryotes Lon is a key enzyme needed for maintaining mitochondrial homeostasis. Despite its important cellular roles, only few native substrate proteins have been identified in most organisms and the mechanisms contributing to its precise regulation remain poorly defined. By focusing our past work on bacterial Lon, our lab has established proteomics approaches that have enabled the identification of large groups of specific Lon substrates in different bacterial species (Omnus & Fink et al. 2021), including regulators of cell cycle progression and cell differentiation as well as stress response proteins (Omnus & Fink et al. 2021, Akar et al. 2023, Kallazhi et al. 2025). Additionally, we have identified and in detail characterized two novel regulators of Lon (LarA and HspQCc) that tune the degradation of specific groups of substrates at the onset of cellular stress (Omnus & Fink et al. 2023, Thulstrup, in preparation). Both regulator proteins allosterically modulate Lon activity by binding a common site in the N-terminal domain (NTD) of Lon, thereby altering substrate selectivity. After having focused for the past 13 years on bacterial Lon, we recently received funding from Cancerfonden to extend our work to human mitochondrial Lon (LONP1). A number of previous studies have associated mitochondrial LONP1 with cancer and other human diseases. Upregulation of LONP1 in cancer cells has been demonstrated to favor tumor growth and metastasis, while reduced Lon activity is thought to promote senescence and cell death. However, the molecular mechanisms underlying LONP1-dependent protein degradation and its connection with human diseases remain poorly understood. Project Goal: This specific project aims to leverage AI tools, mainly Alphafold3, to identify novel protein-protein interacting partners of human mitochondrial LONP1. We hypothesize that regulators functionally similar to LarA and HspQCC exist in mitochondria that modulate LONP1 activity. By conducting a proteome-wide in silico pull-down approach of LONP1 as well as LONP1-NTD in isolation we will generate a list of candidates of LONP1 interactors. Subsequently, we will perform in-depth wet lab experimentation and validations on selected candidates to study the role and relevance of the AI-identified protein-protein interactions. Project Impact: This project will provide novel insights into how mitochondrial LONP1 regulates cell function by identifying previously unknown interacting partners, establishing mechanistic connections between its dysfunction and various diseases. The findings could lead to the development of new therapeutic targets and diagnostic markers for conditions like cancer, aging, and neurodegenerative diseases. Additionally, this work establishes a new experimental pipeline by combining a cutting-edge AlphaFold 3-driven in silico screen with rigorous wet lab validation. Our integrated approach demonstrates the power of AI in biological research and serves as a model for future studies aimed at dissecting complex protein networks.