Interacting proteins of AMPK studied using TurboID proximity labeling technology

Abstract

Abstract Objective Adenosine monophosphate activated protein kinase (AMPK), whose activity is regulated by the AMP/ATP ratio in the body, is an important center for controlling cell energy metabolism, and is also an evolutionarily conservative serine/threonine protein kinase. AMPK plays an important role in diabetes, myocardial infarction and many diseases. We try to use TurboID technology to study a novel protein that can interact with AMPK, and explore the biological function of this protein, in order to provide a theoretical basis for the development of new targeted drugs. Methods We will construct AMPK overexpression stable cell lines by transfecting AMPK-TurboID fusion gene into astrocytes U251 using lentiviral infection technique. After 6 h of biotin labeling, a large number of proteins interacting with AMPK can be observed by silver staining. The interacting proteins were analyzed by label-free quantitative protein profiling, and the interacting protein DNAJA1 was selected for IP and immunofluorescence validation. Results We successfully constructed AMPK-TurboID overexpression stable cell lines, and obtained a large number of interacting proteins after biotin labeling experiments, and then obtained all interacting protein information by mass spectrometry, and selected the interacting protein DNAJA1 for IP and immunofluorescence validation. In addition, we found that AMPK and DNAJA1 could be jointly involved in anti-apoptotic cell death. Conclusion Because AMPK is involved in a variety of metabolic pathways, coupled with the advantages of high catalytic activity and fast labeling of TurboID neighboring labeling technology, a large number of proteins interacting with AMPK gene were found through biotin labeling experiments, and subsequent experiments verified that AMPK and DNAJA1 have interaction, and the two can synergistically protect cells from apoptosis; this has laid a certain theoretical foundation for how to use AMPK to treat clinical diseases such as diabetes and myocardial infarction in the future.