Interacting proteins of AMPK studied using TurboID proximity labeling technology

In living organisms, the change in the adenosine 5'-monophosphate (AMP)/adenosine 5'-triphosphate ratio serves as a key signal regulating AMP-activated protein kinase (AMPK) activity, which is an important molecule for the control of cellular energy metabolism and serves a key role in a number of diseases, such as diabetes and myocardial infarction. To explore novel AMPK-interacting proteins and investigate their biological functions, the present study used TurboID proximity labeling in U251 cells stably expressing AMPK-TurboID. Novel AMPK-interacting proteins were explored under both normal and CCCP-treated conditions to investigate their biological functions. Stable cell lines overexpressing AMPK-TurboID were successfully established, and interacting proteins were identified through biotin labeling, silver staining and mass spectrometry analysis. Due to the involvement of AMPK in numerous metabolic pathways and the advantages of TurboID over traditional techniques, including its high temporal resolution, in situ labeling in living cells and its ability to capture weak and transient interactions, a number of proteins interacting with AMPK were identified. Six proteins identified by label-free MS were validated by western blotting, showing that the expression levels of menage a trois-1 and DNAJ heat shock protein family (Hsp40) member A1 (DNAJA1) closely matched the MS data. DNAJA1 was selected for further experimentation. TurboID proximity labeling, affinity purification and LC-MS/MS, western blotting, co-immunoprecipitation (co-IP) and immunofluorescence (IF) techniques demonstrated that AMPK and DNAJA1 not only interact with each other but also synergistically protect cells from apoptosis, perhaps providing a basis for targeted therapeutics (such as treatments for diabetes and myocardial infarction). The present study achieved covalent labeling of endogenous proteins located within nanometer distance of the labeling enzyme by adding a biotin-based substrate. The labeled proteins were then captured and enriched using streptavidin-coated magnetic beads for subsequent identification by MS. These MS results, combined with subsequent validation, led to the identification of DNAJA1 as a key AMPK-interacting protein that synergizes with AMPK to protect cells from apoptosis.